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MOSQUITO CONTROL

This article is about MOSQUITO control. It will explain why they are a pest and what needs to be done for controlling infestations. PLEASE NOTE: YOU CAN SEE PICTURES AND PRICING OF ALL THE PRODUCTS LISTED IN THIS ARTICLE BY CLICKING YOUR MOUSE CURSOR WHERE PRODUCTS APPEAR UNDERLINED IN THE TEXT BELOW. Most of your questions will be answered in the article. Be sure to read all of it before you call in for technical support. If you are looking for information about any other insect or animal, go to our article archive section by following the link below where you will find in depth articles and information on just about any pest. CLICK HERE TO GO TO OUR ARTICLE SELECTION PAGE Mosquitoes are a pest that are capable of ruining a great day at the park, a romantic evening on a deck or even spending time in the garden. People will do anything to avoid being bit. They will wear long pants during the summer, use hats with screen veils and even spray themselves with everything imaginable hoping that no mosquito will find them. However, mosquito population's are more active today then ever. If you enjoy the great outdoors, get used to dealing with mosquitoes. They are here to stay and this article will explain methods of control you may use to help diminish their numbers around the home. The author will not get in to great detail about all the species throughout the world. Furthermore, this article is not about diseases - specifically the West Nile Virus - nor detailed mosquito biology. There is some basic information the author will discuss but the focus of this article will be to offer control methods for all kinds of mosquitoes - regardless of species!!! There are over 150 species of mosquitoes in the United States. Some are able to fully develop from eggs in less than a week. Most take 10-14 days to reach maturity but what is important is they grow rapidly. Mosquitoes need water and high levels of moisture to sustain themselves. Although female mosquitoes may live for up to a year, most die in the season they were born. Mosquito populations are able to continue from year to year because one stage is able to overwinter and start their cycle again the next spring. It may be the adult, the pupa, the larva or the egg which is needed. Each species has different winter survivors. Some adult females don't need a blood meal to begin to reproduce. In general, male mosquitoes live a short time. Most mosquitoes lay several hundred eggs and are able to generate huge populations within a short period of time. Although standing water is the prime location for them to reproduce, there are many locations around the home that afford fertile egg laying areas. Such places include water in the bottom of planters, drainage streams, street sewers which don't drain completely, rain barrels, buckets of water, swimming pools, drain lines from rain gutters, old tires, mulch around the home, shrubs, trees, firewood, slow moving water, small decorative ponds for pet fish, bird baths, water accumulating around windows or doors, water accumulating from an automatic sprinkler system, pet water dishes, leaks around water spickets and just about anywhere water is used or is able to accumulate during the warm summer months any where in the country. Mosquitoes need water to reproduce. They will readily move to moist, shady areas under decks, around pools, in garages, in dense shrubbery or flowers, any kind of ivy, holes or nooks of trees, water in a clogged rain gutter or simply the water on a leaf of shrubs which are being watered during the hot summer months. Most people believe mosquitoes are coming from great distances to their yard in search of food. In fact, most mosquitoes migrate to a yard first and foremost because there is something about the yard which the mosquito finds attractive for living. In most cases, mosquitoes are finding a great place to live around the home and then take advantage of the free meals the homeowner or their children present when outside in the yard. Mosquitoes don't migrate far from where they will find shelter and protection from the hot sun. Shade and moisture are two ingredients needed for their survival and can be found around any home. If your home is on a lake or pond, the mosquitoes could be breeding in the water. Generally, they will do so close to shore. Don't expect to find them more than 10 feet from shore. They like shallow water and will keep themselves close to plant life and wet lands if possible. Open deep water which is moving is not the kind of water they like for reproduction. Barns or sheds are another great location for reproduction or shelter. The underside of most decks which are built close to the ground offers great shady shelter and protection for weak mosquitoes susceptible to the hot sun. It is important to locate any area around the home where mosquitoes may be seeking shelter or using for reproduction. The author has experienced a trend in recent years which will only continue. Many homeowners are creating perfect breeding and shelter conditions which are attracting mosquitoes. If you have any of the conditions described above, chances are you will have mosquitoes. Don't be placing the blame on someone else. Mosquitoes will stay where the breeding and shelter areas are best for them. If you are creating a moist shady area around your home, you will be luring mosquitoes. Once they find the shade and moisture to live, expect them to find you and your family for their food! Mosquito control is easy if you are able to determine where they are living or breeding. Inspect around your home and locate where the mosquitoes are most prevalent. Although you may believe they are coming from an adjacent lot, be sure to inspect your property thoroughly. If you have any of the sights listed above, chances are mosquitoes are taking advantage of such conditions. Another way to determine where the mosquitoes are living is to simply stand in certain areas and wait to see how long it takes for them to find you. Mosquitoes will not travel far away from where they are comfortable. The faster you have mosquitoes find you and the more that find you indicate a prime nest or shelter location which needs to be treated. Such "pockets" of mosquitoes exist around most any home and the secret of getting control of the situation is to find as many of these locations. Once found, there are several methods of treatment that can be used. Now that you have determined where to treat, you will need to choose the product best suited for your situation. Mosquito products are designed for treating different types of areas. There are five different product types. First, there are repellents. These are used for repelling mosquitoes from any treated area or from people themselves. Second, there are larvacides. These are products which kill the larva of mosquitoes as they develop. They are usually applied to water where mosquitoes are breeding. Third, there are residual products. These are insecticides which can be applied with a standard pump sprayer or a garden hose end sprayer. Applied to the surfaces where mosquitoes like to land, the residual of these materials will kill mosquitoes as they enter and land on treated surfaces. The fourth type of product used for mosquito control is to directly kill them as they fly. This can be done by aggressively fogging for them or by passively trapping and killing them. Fogging machines offer the most immediate and complete control of all options whereas the newer Mosquito Killing Systems are passive yet somewhat effective. The fifth type of mosquito control that can be employed is a relatively new approach yet one which is both easy and effective. Much like many of the new allergy medicines, there are now Mosquito Blockers. These are devices which affect mosquitoes in such a way they are not able to detect Co2 or octenol. Not being able to detect these compounds prevents them from being able to track a target. Lets examine the products in each category and decide so that you can decide which one will work best for your problem. Repelling mosquitoes have long been the most common method of trying to control local infestations. In fact, there was no real "control" going on; repellents simply push the pest to another area. Citronella, smoke and other compounds have been used over the years but all met with little if any success. However, we now have several repellents that work OK for certain types of applications. The key here is the type of repelling you are looking to achieve. Don't think you will be able to keep mosquitoes away from your yard if they are reproducing or nesting on or adjacent to your land. You don't stand much of a chance for long term repelling under these conditions (as described above). However, you can achieve some temporary relief with these products. They will help to reduce activity around your patio or picnic area so that you can better enjoy the outdoors for a picnic or summer party. MOSQUITO REPELLENT POWDER is a powdery material which can be applied over the turf and plant area where you want to keep mosquitoes away. It is easy to use, will last a week or more (under dry conditions) and poses no hazard to people or animals. In fact, it uses a high PH carrier which conditions the soil much like lime. This product offers short term relief and should not be used with the intention of "controlling" mosquito infestations. Mosquito Repellent is designed to be used right before a party or outdoor get together where a reduction in mosquito activity is desirable. Use some INSECT INCENSE directly around the area as well to aid in the results you will be able to achieve. They are pleasant smelling and non-offensive to people yet mosquitoes and other biting insects don't seem to like being around where they are burning.If you want something a little stronger, set out some of the MOSQUITO COILS. These are not the Citronella type you may have seen in the past. These are the newer formulated type which have a Synthetic Pyrethrin as the active ingredient - a known repellent to flying insects. Though there may be some which enter the perimeter defense, the Grids or Coils are then there to help prevent them from being able to find their meal! These products are great for around the home and the Grids or Coils are certainly portable enough to bring camping, but how can you repel mosquitoes when you are golfing, fishing or doing other outdoor activities? There are two other types of repellents which can help. The first one is the BUG BAND. This is worn around the wrist and like the Grids, releases a scent which actively confuses mosquitoes and prevents them from identifying you as a target. These will last many hours, can be stored for long term reuse and come in both adult and children sizes. The latest of the personal repellers which works well is the PERSONAL BITE SHIELD. This unit is made of a strong durable plastic and features a small fan which is powered by two AA batteries. The unit holds repellent cartridges which have the latest mosquito repellent, Geraniol, which is all natural. Geraniol is extracted from plants where it serves as a natural repellent plants use to keep predatory insects away. Each cartridge is snapped onto the Shield and the fan is turned on which disperses the Geraniol around the person wearing the device. It's about the size of a pager, fits on your belt or can be set on a table, ledge or other area where you are sitting. The pleasant light odor released will keep mosquitoes at bay naturally and it can even be hooked up to an external power supply. If you want something for your skin, which is the most direct way of repelling mosquitoes, consider either CITRONELLA LOTION or DEET. Both will repel mosquitoes as well as other biting insects. Citronella is not nearly as strong as the Deet and expect to apply it several times a day. Deet will last much longer and do a better job overall at keeping most any type of pest away. It comes in different strengths and flavors with the 100% form being a low odor mix. For complete control when traveling abroad or into areas where mosquitoes pose a real health risk, use some PERMETHRIN to treat clothing, gear and shoes. Permethrin is odorless, labeled for many uses and will keep mosquitoes away since it acts as a detectable repellent they don't like. It is important to understand that the products listed above are not "controlling" local infestations. They should be used when other methods of true control are not an option or if you only want some temporary relief. Repellents of today are better than ever; however, they do just that - repel. You may want to keep a jar of MOSQUITO OINTMENT handy for when you end up getting bit. This soothing treatment takes the itch away and promotes fast healing. Small enough to take out into the field and since only a little is needed, it goes a long way. Another option is to keep them off you all together when going afield. This can be done with our MOSQUITO HEAD NETS. These are lightweight and come with elastic bands. Simply pull them over your head and they will fit snug enough to keep mosquitoes off your face but loose enough so you will still be comfortable. These are an excellent item to bring along when fishing or camping and the pressure from mosquitoes or other biting pests is simply too great. The screen is pre-treated with Permethrin so it offers some repellency as well. If you want no chemicals on it simply wash it once and all the Permethrin will be gone. However, we suggest spraying it periodically with our Permethrin Aerosol featured above. This treatment will help to keep them away from you all together adding that extra protection sometimes needed. We also have four types of netting which can be used for sleeping. These include the TRACKER NETTING, the SINGLE BED NETTING, the DOUBLE BED NETTING and the CANOPY NETTING. All are made from the same light screening which lets air flow unrestricted but is sure to keep out any biting insect. All come with rope allowing you to hang them from a tree or ceiling; all are pre-treated with Permethrin. The Tracker is designed for campers. It comes with it's own storage pouch making it easy to carry along in any backpack. It won't take up a lot of space but it will certainly make the trip a lot more enjoyable. The Single Bed Netting is for just that - a single bed. Use it where windows are kept open inviting night time biting insects. The Double Bed Netting is the same look and design. It's just large enough for a double bed. The Canopy Bed Netting is the more elaborate and complete design which can be used to elegantly dress up any bed yet provide protection from flying pests. It features an entrance which overlaps which is fashionable as well as usable. Though the Tracker is best suited to go afield, any of them can be brought with you as you travel. The Single and Double Bed Netting come in a simple heavy plastic bag in which to store them when not in use; the Canopy Bed Netting comes in a white burlap bag. MOSQUITO DUNKS look like a donut and are used in water where mosquitoes are reproducing. The dunk will slowly melt away releasing thousands of bacteria which will kill mosquito larva. By killing the larva, the mosquito reproduction will stop. This has long been the approach people have used when controlling mosquito populations. The dunks are so safe they can be used in ponds, bird baths and water holding tanks without being a hazard to pets, wildlife or people. They can be used in retention ponds, catch basins for plants and drainage ditches. Use them anywhere you know that water will be held for 3 weeks or more. Because the bacteria is simply digested by mammals (which includes pets, people and wildlife), there is no hazard to this product being used in water used for drinking. If you have water which will only be available for 1-3 weeks, you can still treat with Mosquito Dunks but MOSQUITO GRANULES will probably work better. Because they release quicker, they will impact the developing mosquitoes that much faster. Mosquito Granules have a short life; you may need to treat once a week since they break down so fast. However, they are perfect for small areas such as plant catch pans, bird baths and rain barrels. Made from the same bacteria as the original dunk, Mosquito Granules are able to stop developing mosquitoes from reaching adulthood. If you want something like the Mosquito Granules but a little stronger, get the METHOPRENE GRANULES. These look like the other granules but use a growth regulator instead of just a bacteria to impact growing mosquitoes. The active ingredient in this granule is Methoprene. Long used for flea control, Methoprene will stop mosquitoes from being able to mature to reproducing adults. Since Methoprene is essentially a protein, this product is still very safe to use and is labeled for all the same areas as the other granules. However, there is no hit or miss with this product. Any mosquito larva that are in treated water won't be able to grow right or fully mature so this is great to use in ditches, free standing water, moist and wet compost or flower beds, water gardens, tree holes, roof gutters, pool covers and just about anywhere water is able to pool. The other great thing about this form is that a little goes a long way making it much more practical to use. You will have to apply it at least once a month but regular applications will prevent local activity from completing their life cycle. If you have a lot of plant life and landscape around the home that requires water throughout the growing season, chances are you will attract mosquitoes. Shrubs, annual flowers, thick Fescue and Bermuda grass or ivy all provide pockets of moisture where water can last and provide shelter for mosquitoes. As warm and hot summer months dry local wet lands, expect mosquitoes to migrate in search of moist, shady areas. Many pool owners or homes with decks and porches provide perfect conditions for mosquitoes. The shade and moist areas are what mosquitoes need to survive and you will notice populations increasing as it the hot summer sun diminishes local watering holes. Use a residual product to treat these areas. ESFENVALERATE is a great product to use when treating these areas. It is easy to apply, is labeled for use on turf, trees, shrubs and flowers and will provide residual for long term control. Mosquitoes are easy to kill. Esfenvalerate will last several weeks on treated surfaces and does very well under decks, porches and other shady areas mosquitoes are attracted to. Use a DIAL-A-MIX applicator which hooks to your garden hose to apply Esfenvalerate as it requires a lot of water. It is used at a low rate so a little bit goes a long way. You will see immediate results following the initial treatment. It is hard to say how long you can expect to be mosquito free. Every area will experience different results based on weather, moisture, wind, local breeding conditions, rain, temperature and humidity. Expect to get 1-2 weeks of protection per treatment. Dry environments can expect longer lengths of residual. Retreat as necessary. If you are going to be doing some residual spraying with the Esfenvalerate, a good idea is to add a growth regulator to the mix. This will enable you to get long term control over the areas being treated. Long used for flea control, growth regulators have now found a niche for controlling mosquitoes. Earlier in this article, the Methoprene Granules were highlighted. They use a growth regulator and are designed to be applied in water. Another growth regulator that is designed to be sprayed out is NYLAR. This product is newer then Methoprene but works much the same way. Basically it interferes with the mosquito larva's ability to grow properly so they are not able to fully develop to biting adults. Use it with the Esfenvalerate since Nylar won't kill any adults; it only works on eggs and larva stages. Though you can use a standard pump sprayer to apply the two materials, if you have larger areas to treat, the Dial-A-Mix Sprayer featured above will better serve you. Just add 1 oz of the Nylar with 1 oz of the Esfenvalerate for every 1500 sq/ft of area you want to treat and set the dial to 4 for a good spray mix. Adding the Nylar to the tank mix really makes sense and will cut down on the frequency of treatments needed by about 75%. In most cases, Esfenvalerate by itself will have to sprayed at least twice a month. When used with the Nylar, you will only have to treat once every 2 months. Combining the two products will cost more at first but in the long run will end up saving you a lot of work, a lot of time and a lot of cost. Space spraying has long been the standard way to treat for mosquitoes and may be necessary in certain areas. Since mosquitoes are able to fly, they may be coming to your yard for food and living directly alongside your land in wet, swampy conditions that offer shade and moisture. Such conditions, when large, are hard to treat with Esfenvalerate as described above. To penetrate dense foliage or to cover large areas quickly, space spraying is the preferred method of treatment. You may be able to treat small areas like this with an aerosol like PT-565XLO. This product uses pyrethrum as the active ingredient and will kill mosquitoes quickly. Spray it around decks and picnic areas to provide yourself with a few hours of relief. It is easy to use and is great for around the garden, deck, patio or back yard area. AEROSOL MACHINES are also good for small areas. They can be hung on the wall, are battery operated and can be programmed to release a blast of pyrethrin based aerosol which works well on flying insects. Use some METERED AEROSOL in these machines and they will keep most any flying pest under control. Use of these products is for small areas only. If you are treating a larger area, use an electrical fogging machine such as a FM6309. These machines are able to pump a lot of chemical over a large area in a short amount of time. They create a "fog" or "mist" which floats around plant leaves and other areas mosquitoes like to hide. This machine produces a cold fog which is adequate for most home owner applications. It is able to treat 1/4 to 1/2 acre in less than 30 minutes. (The machine will pump a gallon of material in around 12 minutes, but it takes extra time to drag the extension cord to new treatment areas.) If you have a lot of small areas to treat, you may prefer the FM6208. This fogger is a step up from the FM6309 because it has a volume control switch which allows you to adjust the rate of flow. It still uses electricity to power it, but unlike the FM6309 it has a rotation switch which can be set to off, low, medium or high. This can be a real help for two reasons. First, you are able to turn it keep it low when treating around the home and you only need a light mist. Such treatments can be tough with the FM6309 because it only fogs at one speed - high! When you try to treat alongside a structure, the fog is so strong it will bounce off the building and come back at you. If you have to do a lot of this type of fogging, get the FM6208. It not only lets you turn it down for these types of delicate applications but the rotating switch will let you turn it to off. Though the machine is still on and trying to fog, it will quickly run dry. This is handy if you are interrupted during treatment. Simply turn the switch to off and let the fogger use up what's in it's system. Now you can store it for a few days without worrying about the insides getting clogged. Two other models you may want to consider are the FM7807 and the MINI FOGGER. The FM7807 features the same controls as the FM6208 but also has a long extension. This extension is great for reaching high, under and around things which other machines have difficulty covering. The FM7807 also features a quick on/off control on the wand; great for when you need precise control of flow. The Mini Fogger is basically a compact version designed for the person who has only a very small area to treat. It can cover areas from 2500-5000 sq/ft very well. It uses electricity, has a volume control and a quart holding tank. Great for the condo or townhouse owner who has a small area to treat. All these foggers are great for jobs around the home but they do require electricity and are not nearly as portable as the BACKPACK FOGGER. This device has over a two gallon tank and is gas powered. Perfect for those large jobs where electricity is not available. Simply fill up the holding tank, make sure you have enough gas in the tank and you can go wherever the mosquitoes may be hiding! Great for property where being free from electric cords is necessary or if you have a lot of areas to treat on a regular basis but need to move from one point to another as you treat. Once you decide on which type of fogger will fits your needs, you will have to get some chemical. Use water as the carrier for the tank mix and try to reach as much as the shady, undercover where mosquitoes tend to hide. Though products like Malathion have been used over the years for such applications, you will achieve better results with Pyrethrum and Permethrin and they have little if any odor. PYRETHRUM is natures own pesticide and is very active on mosquitoes. It will kill adults on contact and works on many other flying insects as well. As good as Pyrethrum is, it will not provide any residual. Mix PERMETRHIN in the tank for better results. Permethrin does not provide the quick kill like Pyrethrum but it does last from 1-2 weeks. This will prove helpful when treating large areas. The residual will help to stop the mosquitoes from reproducing or from providing shelter to wanderers that like the moist shady shelter a large wooded area adjacent to your home may provide. The one-two punch of pyrethrum and permethrin will kill existing populations of mosquitoes in hard to treat vegetation and keep new infestations from being able to form. And though this combination is hard to beat, the addition of some NYLAR to the mix could help cut down on the frequency of treatments needed throughout the season. As explained above, Nylar works on eggs and larval stages of mosquitoes and by doing so will prevent their young from reaching maturity. This "breaking of the "cycle" means that fewer treatments will be needed over the course of the season and by having this impact, the amount of time and chemical used should be decreased as well. If you have larger areas to fog than an acre, you should consider moving up to a thermal fogger. Thermal foggers are "hot"; that is, they heat the chemical before it is released. This type of fog is lighter and comprised of smaller sized particles than that of a cold fog. Thermal foggers are more thorough during application and require an oil based formulation instead of water. The fog will stay in the air longer and penetrate more providing better performance than fogs created by electric machines. However, there are some tradeoff for this better performance. First, machines are costly. Don't waste you time with $100.00 to $200.00 units found at some hardware stores. These devices will create some fog, but the flow is so weak and slow you are better off using the PT-565XLO listed above. If you have anything larger than a few thousand feet to treat, these cheap machines are just not able to do the job adequately. If you have a large area that you want to keep mosquito free, invest your money with a machine that will do the job. The GE THERMAL FOGGER is gas powered, electric start and lightweight. It can be carried easily to treat large areas or you can let it run out the back of your ATV or truck as you drive through the property you are treating. This machine will create a fog which is light, penetrating and very effective for mosquito control. Be sure to use the proper material when fogging to achieve the best results. Oil based PYRETHRUM or SYNTHETIC PYRETHRUM are needed. These formulations will provide quick knockdown of existing populations and you can add some Permethrin to the mix as detailed above to get extra residual. If you intend on doing a lot of fogging, you should consider getting our DEODORIZED OIL in bulk form and then adding Pyrethrum and Permethrin to the mix. This will prove to be the most economical formulation available when treating large areas many times during the season. Like fogging with electric foggers you may need to treat twice a week to get control of bad problem areas, but once you knock down the local population you will probably only have to treat once every week or two. Another option that needs to be mentioned is the installation of a Mosquito Killing Machine. There are several being made these days and most any retailer has one style or another. These machines all use some type of attractant to lure mosquitoes to them. It could be CO2, heat, moisture or something else that biting insects find attractive. Once lured to the device, most have a mechanism that will somehow either kill or remove the pest. The goal of these machines is to remove as many pests as possible in your yard. Though most will kill a lot of insects, it is not likely that any will adequately remove enough to have the desired result. We have carried many different such devices over the years and though several have proven to do a good job of killing mosquitoes, none have proven to keep a designated outside area mosquito free. Since these applications only kill adults and do nothing to address the reproduction sites, larval stages and other key components of the mosquitoes life cycle, they don't have nearly enough of an impact to warrant implementation. Since most people purchase these units with the hopes that they won't get bit anymore, we have decided to drop all but one model at this time. The model we continue to carry is mostly designed for inside applications but can be used for small decks, enclosed patios or screened in rooms. Such areas usually only have a few flying insect problems and this machine will do a good job of killing off any biting pests which may be around. For larger areas, use one of our foggers or liquid materials for the best control. But if you have a room or two in the home which seems to get a lot of mosquitoes or other biting pests, install one of our INDOOR MOSQUITO TRAPS. It has several attractants that do a good job of attracting local mosquitoes and can handle small confined areas well. Don't get it with the hopes that you will never see another mosquito or biting pest; such results are not likely. However, they will help and general rule when treating for mosquitoes is that the more materials being used, the better. Another type of trap which is relatively new but will prove to be quite helpful when dealing with mosquitoes is called the MOSQUITO PHEROMONE TRAP. This trap is made of a small plastic jar which is about 1/2 gallon in size. The jar is filled with one quart of water and some "vegetation tablets". These tablets are basically plants which have been freeze dried and once mixed with water will convert back to their old decaying natural state. Such areas female mosquitoes will seek out as good egg laying locations.Be sure to used distilled water or let a quart of water sit overnight so the chlorine level dissipates. Chlorine will disrupt the natural decaying process and prevent certain odors from releasing. This will interfere with the mosquitoes ability to find the trap. Add the water to the jar along with the pellets and then about 3 drops of dish detergent. The jar has a small tube that runs through its middle which serves as an entrance as well as a holding area for a EGG LAYING PHEROMONE which is then set out with the trap. Be careful not to touch the pheromone; human scent, natural oil from our fingers or some other contaminate will erode its effectiveness. The pheromone is a scent which mosquitoes naturally release in the wild when they find a good egg laying location. This "locator pheromone" is then smelled by local mosquitoes ready to lay eggs and will attract them inside. Once inside, they will attempt to land on the water. At that point, the detergent which was added to the tank will cause them to sink into the water and drown! This will happen over and over during the course of the season which can have an astounding impact on local populations. Try to place out 2-4 traps for the average yard; about 6 per 1/2 acre. The pheromone should be replaced every month and be sure the water level does not drop too low during the hot and dry season. Also, be sure to keep Traps along property borders or where people won't be active; preferably in moist shady areas. You can also equip yourself with a HAND HELD ZAPPER. This is really handy when dealing with mosquitoes which are sometimes hard to swat. Simply wave the Zapper where the mosquito is flying in a gentle manner and once it makes contact with the Zappers grid it will be dead. Don't try to swat them, this Zapper is not designed for such use. However, it is a great tool to keep at your side when mosquitoes are in the area and you want an easy and clean way to kill them. And since mosquito's are so slow when flying, you can even catch them with our BUG VACUUM/ZAPPER cordless tool. This device is generally best for crawling insects but works well for mosquitoes and just about any flying pest which likes to land on walls, windows or ceilings. Though effective at killing many mosquitoes, don't be lured into using these devices because it is so easy to install and operate. It is another tool and when dealing with mosquitoes, the best approach is to incorporate as many tools as is needed. You will get much better results if you treat property with Esfenvalerate or Fog and install a Mosquito Killer. The final method of keeping mosquitoes away can be done with a Mosquito Blocker. This is relatively new technology but the logic behind it makes a lot of sense. It's kind of like the new allergy medicines. Most allergy medicine is designed to deal with the symptoms of the allergy - the itchy eyes, running nose and congestion. Newer medications are histamine blockers which essentially block the uncomfortable symptoms of having allergies. You still have the allergy - just not the symptom. This approach is very effective, relatively easy to administer and tends to be healthier in the long run. The same is true for the Mosquito Blockers. These devices are small plastic machines which run on 2 AA batteries. They will operate at least 720 hours and are used to power a small fan. The fan blows out a product called Conceal. This is not a pesticide. Conceal is comprised of plant oils and other natural ingredients which bind to insect olfactory sensors. The impact of Conceal is that mosquitoes and several other biting insects are not able to detect Co2 or octenol - two gases which people (mammals) exhale all the time. By not being able to detect the presence of Co2 or octenol mosquitoes further than 30 feet away will not be able to track your presence. You will become invisible to them. Remember, mosquitoes have a limited range of sight. It is probably not more than 15-20 feet and certainly not more than 30 feet. Mosquitoes which are not able to see any targets will then begin to trail or track their prey by detecting Co2 and octenol. As they fly around they are constantly hoping to find some of these gases in the air. Any breeze will carry your exhaled breath several hundred feet and mosquitoes along this path will be able to trace it back to it's source - YOU! By installing a MOSQUITO BLOCKER about 10-20 feet downwind of where you are sitting, the Conceal will be carried with your exhaled gases. As mosquitoes cross this path they will be affected by the Conceal in such a way that they will not be able to detect the Co2 or octenol. These units work best when there is a slight breeze. Simply place a unit close by, downwind, and go about your activities. You won't even know it is there after a few days. It's perfect for the front porch, decks, sitting areas out in the yard, pool or pond and is completely portable. Just remember to bring it back inside and to keep a fresh supply of CONCEAL in it to enable to work properly. If there is no wind or breeze present, you will probably need to locate two or three machines around you. Remember, the more the better. If there is no real direction to the way the air is moving then try to keep one on any side mosquitoes can approach. This may mean having 2,3 or even 4 machines. As a general rule you need to have more machines as the number of people increases as well. This is due to the increase of gases being released. The Mosquito Blockers are an excellent tool which can be set out easily and will help provide additional relief from the pressure local mosquitoes will apply. When used in conjunction with Mosquito Killers or Foggers you can really reduce the amount of bites you, family and friends have to endure. Mosquitoes have long been an enemy of man and our lifestyle. To enjoy the great outdoors, you must first learn why they are attracted to your land. Once shelter and breeding locations have been identified, choose the best approach to stopping them. Though the Bug Bands and Deet will help if you are out in the field, treatments with our Repellent Granules, Dunks, Esfenvalerate or Fogging Compounds will be necessary for complete and pest free areas around the home. Be sure to get enough "machine" when fogging so the time you spend treating will be minimal and the time you are able to enjoy the great outdoors is maximized. Set out some Blockers for added protection and you just might be able to enjoy the great outdoors once again! To see any of the products listed above, click on their name as it appears in a different color or underlined. The link will bring you to our product catalog where you will be able to see the product and learn more about it. You can also link to our product catalog below. Check out the rest of our catalog and make sure to keep us bookmarked! We've got the products for whatever is bugging you! CLICK HERE TO GO TO OUR MOSQUITO PRODUCTS CLICK HERE TO GO TO OUR PRODUCT SELECTION PAGE CLICK HERE TO GO BACK TO OUR ARTICLE SELECTION PAGE CLICK HERE TO GO TO OUR MAIN PAGE Our toll free number is 1-800-877-7290. E-Mail us at support@bugspray.com All articles copy righted by U-Spray, Inc. 4653 Highway 78 Lilburn, Georgia 30047 Phone: (770)985-9388 Fax: (770)985-9319 Toll Free: 1-800-877-7290 url: http://www.bugspray.com/articles98/mosquitoes.html

HOME REMEDIES, HOLISTIC APPROACH, & REPELLENT PLANTS - Chinaroad Lowchens of Australia

HOME REMEDIES, HOLISTIC APPROACH, & REPELLENT PLANTS - Chinaroad Lowchens of Australia

Some suggested old home remedies. No endorsement is intended nor liability assumed since most of these home remedies are not proven or approved as pest control recommendations.

1.

Banish ants from your pet's food dish by wiping the floor under and around it with a cloth dipped in kerosene. Then stand the food dish in a larger dish containing water.
2.

Keep ants from crawling up a picnic table by standing each leg in a small pan of water.
3.

To kill ants, use a paste of equal parts of borax and confectioner sugar.
4.

Mix mint apple jelly and boric acid for ant control (two tablespoons boric acid powder per 10 ounces of mint apple jelly).
5.

Leave a few tea bags of mint tea near areas where the ants seem most active. Dry, crushed mint leaves or cloves also work as ant deterrents.
6.

Keep a small spray bottle handy, and spray the ants with a bit of soapy water.
7.

Mix peanut butter (six parts), brown sugar (one part), one-half teaspoon salt with boric acid (one part) for ant control.
8.

For ant control, spray vinegar around door and window frame, under appliances, and along other known ant trails.
9.

If ants are coming in through doors or windows, put a cinnamon stick across the path. They will not cross it.
10.

Mix three cups water, one cup sugar and four teaspoons boric acid powder for ant control. (Pour a over a cotton wad in a small dish or bottle cap.)
11.

Sliced or crushed cucumbers to keep cockroaches away from food.
12.

Mix equal parts of boric acid powder, powdered sugar, and cornmeal as a poison bait for cockroaches.
13.

Mix equal parts of plaster of Paris and powdered sugar as a poison bait for cockroaches.
14.

It is a little known fact that roaches like high places. If you put borax on TOP of your kitchen cabinets (not inside), if space allows between ceiling and cabinets, the roaches will take the borax to their nests, killing all of them.
15.

Keep a spray bottle of soapy water on hand. Spraying roaches directly with soapy water will kill them.
16.

Walk through a room wearing white socks to detect fleas. Dark fleas jumping on the white background are easily seen.
17.

Use banana peels to repel fleas.
18.

Feed yeast to dogs to repel fleas.
19.

Fleas HATE Stash Earl Grey. Tear open a few bags, scatter the tea about on your carpet and vacuum up in a few days.
20.

For flea control, add a little vinegar to your pet's drinking water to fight fleas and mange.
21.

For flea control add ½ teaspoon to the wash water or a few drops to the pets shampoo.
22.

Suspend a light bulb over a pan of oil or soapy water to attract and drown fleas during the night.
23.

For a fly repellent - 2 cups vinegar, 1 cup Avon Skin So Soft, 1 cup water, 1 tablespoon Eucalyptus oil, 1 tablespoon citronella oil - Put in a spray bottle and spray dog's coat.
24.

Mix water with cornstarch into a paste and apply. This is effective in drawing out the poisons of most insect bites and is also an effective remedy for diaper rash.
25.

Rub jewel weed on mosquito bites and poison ivy to control itch.
26.

For mosquito bites apply lime juice diluted with water on bites with cotton ball.
27.

Mozzies won't bite if you mix 4 parts glycerine, 4 parts alcohol, 1 part eucalyptus oil. Or make a solution of equal parts of isopropyl alcohol and methyl phthalate.
28.

If you're using the barbeque, throw a bit of sage or rosemary on the coals to repel mosquitos.
29.

Put an opened bottle of Oil of Pennyroyal Essence in the room you want mosquito free.
30.

Use Avon's Skin-So-Soft as an insect repellent for people and pets (good mosquito repellent). It helps relieve itching caused by insect bites and dry skin. Also, mix five parts water, one part Skin-So-Soft and mist on show animals. Brush in to make their coats gleam and keep insects off so your animal won't fidget.
31.

Use hedge apples for control of crickets and spiders.
32.

For grass and weeds growing between stones or bricks on walks or terraces, sprinkle 20 Mule Team borax powder and sweep into cracks (one application every other year).
33.

Apply tobacco and snuff juice for wasp stings and bites.
34.

Use beer or yeast dissolved in water in pit fall traps (cups sunk into the ground) to attract and drown snails and slugs.
35.

Prevent mosquitoes from breeding in rain barrels by applying 1 tablespoon of olive oil to the water's surface.
36.

Tick and Fly Spray - two cups white vinegar, one cup Avon Skin-So-Soft bath oil, one cup water, one tablespoon eucalyptus oil (available at drugstores and health food stores).
37.

Pour hot boiling water and a strong cleaning detergent down the drain to eliminate nuisance gnats and flies.

PLANTS & HERBS

* Basil - plant close to the house and it will repel mosquitos.
* Lavender - plant around the house to repel flies and mosquitos.
* Castor Bean Plant - plant in pots within the house; replant outdoors to repel mozzies.
* Scented Geraniums - plant in pots and the garden to repel mosquitos
* Citrosa Plants - plant in pots and the garden to repel mosquitos
* Lemon Thyme - plant in pots to repel mosquitos
* Citronella Grass - plant in pots to repel mosquitos
* Common Marigold - plant in pots and the garden to repel mosquitos
* Thai Lemon Grass - (Cymbopogon citratus) is an effective mosquito repellent.
* Rosemary - powdered Rosemary leaves are used as a flea and tick repellent
* Chamomile - repels mosquitos
* Citriodora - repels mosquitos

MORE USEFUL PLANTS FOR YOUR GARDEN

* Red Raspberry Leaves (Rubus idacus) - the leaves strengthen the muscles of the uterus
* Plantain (Plantago lanceolata) - leaves used whole or in powdered form for bleeding cuts
* Hen-and-chicks (houseleek) - Sempervivum tetorum - soothes minor stings and burns
* St. John's Wort (Hypericum perforatum) - used for burns and scrapes
* Yarrow (achillea millefolium) - stops bleeding

OFF THE SHELF LOW COST PEST CONTROL

Introduction to Fluorescent Proteins

Nikon MicroscopyU: Introduction to Fluorescent Proteins

ntroduction to Fluorescent Proteins

The discovery of green fluorescent protein in the early 1960s ultimately heralded a new era in cell biology by enabling investigators to apply molecular cloning methods, fusing the fluorophore moiety to a wide variety of protein and enzyme targets, in order to monitor cellular processes in living systems using optical microscopy and related methodology. When coupled to recent technical advances in widefield fluorescence and confocal microscopy, including ultrafast low light level digital cameras and multitracking laser control systems, the green fluorescent protein and its color-shifted genetic derivatives have demonstrated invaluable service in many thousands of live-cell imaging experiments.

Osamu Shimomura and Frank Johnson, working at the Friday Harbor Laboratories of the University of Washington in 1961, first isolated a calcium-dependent bioluminescent protein from the Aequorea victoria jellyfish, which they named aequorin. During the isolation procedure, a second protein was observed that lacked the blue-emitting bioluminescent properties of aequorin, but was able to produce green fluorescence when illuminated with ultraviolet light. Due to this property, the protein was eventually christened with the unceremonious name of green fluorescent protein (GFP). Over the next two decades, researchers determined that aequorin and the green fluorescent protein work together in the light organs of the jellyfish to convert calcium-induced luminescent signals into the green fluorescence characteristic of the species.

Although the gene for green fluorescent protein was first cloned in 1992, the significant potential as a molecular probe was not realized until several years later when fusion products were used to track gene expression in bacteria and nematodes. Since these early studies, green fluorescent protein has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors that are broadly referred to as fluorescent proteins. More recently, fluorescent proteins from other species have been identified and isolated, resulting in further expansion of the color palette. With the rapid evolution of fluorescent protein technology, the utility of this genetically encoded fluorophore for a wide spectrum of applications beyond the simple tracking of tagged biomolecules in living cells is now becoming fully appreciated.

Illustrated in Figure 1 are two examples of multiple fluorescent protein labeling in living cells using fusion products targeted at sub-cellular (organelle) locations. The opossum kidney cortex proximal tubule epithelial cell (OK line) presented in Figure 1(a) was transfected with a cocktail of fluorescent protein variants fused to peptide signals that mediate transport to either the nucleus (enhanced cyan fluorescent protein; ECFP), the mitochondria (DsRed fluorescent protein; DsRed2FP), or the microtubule network (enhanced green fluorescent protein; EGFP). A similar specimen consisting of human cervical adenocarcinoma epithelial cells (HeLa line) is depicted in Figure 1(b). The HeLa cells were co-transfected with sub-cellular localization vectors fused to enhanced cyan and yellow (EYFP) fluorescent protein coding sequences (Golgi complex and the nucleus, respectively), as well as a variant of the Discosoma striata marine anemone fluorescent protein, DsRed2FP, targeting the mitochondrial network.

Green fluorescent protein, and its mutated allelic forms, blue, cyan, and yellow fluorescent proteins are used to construct fluorescent chimeric proteins that can be expressed in living cells, tissues, and entire organisms, after transfection with the engineered vectors. Red fluorescent proteins have been isolated from other species, including coral reef organisms, and are similarly useful. The fluorescent protein technique avoids the problem of purifying, tagging, and introducing labeled proteins into cells or the task of producing specific antibodies for surface or internal antigens.

Properties and Modifications of Aequorea victoria Green Fluorescent Protein

Among the most important aspects of the green fluorescent protein to appreciate is that the entire 27 kiloDalton native peptide structure is essential to the development and maintenance of its fluorescence. It is remarkable that the principle fluorophore is derived from a triplet of adjacent amino acids: the serine, tyrosine, and glycine residues at locations 65, 66, and 67 (referred to as Ser65, Tyr66, and Gly67; see Figure 2). Although this simple amino acid motif is commonly found throughout nature, it does not generally result in fluorescence. What is unique to the fluorescent protein is that the location of this peptide triplet resides in the center of a remarkably stable barrel structure consisting of 11 beta-sheets folded into a tube.

Within the hydrophobic environment in the center of the green fluorescent protein, a reaction occurs between the carboxyl carbon of Ser65 and the amino nitrogen of Gly67 that results in the formation of an imidazolin-5-one heterocyclic nitrogen ring system (as illustrated in Figure 2). Further oxidation results in conjugation of the imidazoline ring with Tyr66 and maturation of a fluorescent species. It is important to note that the native green fluorescent protein fluorophore exists in two states. A protonated form, the predominant state, has an excitation maximum at 395 nanometers, and a less prevalent, unprotonated form that absorbs at approximately 475 nanometers. Regardless of the excitation wavelength, however, fluorescence emission has a maximum peak wavelength at 507 nanometers, although the peak is broad and not well defined.

Two predominant features of the fluorescent protein fluorophore have important implications for its utility as a probe. First, the photophysical properties of green fluorescent protein as a fluorophore are quite complex and thus, the molecule can accommodate a considerable amount of modification. Many studies have focused on fine-tuning the fluorescence of native green fluorescent protein to provide a broad range of molecular probes, but the more significant and vast potential of employing the protein as a starting material for constructing advanced fluorophores cannot be understated. The second important feature of green fluorescent protein is that fluorescence is highly dependent on the molecular structure surrounding the tripeptide fluorophore.

Denaturation of green fluorescent protein destroys fluorescence, as might be expected, and mutations to residues surrounding the tripeptide fluorophore can dramatically alter the fluorescence properties. The packing of amino acid residues inside the beta barrel is extremely stable, which results in a very high fluorescence quantum yield (up to 80 percent). This tight protein structure also confers resistance to fluorescence variations due to fluctuations in pH, temperature, and denaturants such as urea. The high level of stability is generally altered in a negative manner by mutations in green fluorescent protein that perturb fluorescence, resulting in a reduction of quantum yield and greater environmental sensitivity. Although several of these defects can be overcome by additional mutations, derivative fluorescent proteins are generally more sensitive to the environment than the native species. These limitations should be seriously considered when designing experiments with genetic variants.

In order to adapt fluorescent proteins for use in mammalian systems, several basic modifications of the wild-type green fluorescent protein were undertaken and are now found in all commonly used variants. The first step was to optimize the maturation of fluorescence to a 37-degree Celsius environment. Maturation of the wild-type fluorophore is quite efficient at 28 degrees, but increasing the temperature to 37 degrees substantially reduces overall maturation and results in decreased fluorescence. Mutation of the phenylalanine residue at position 64 (Phe64) to leucine results in improved maturation of fluorescence at 37 degrees, which is at least equivalent to that observed at 28 degrees. This mutation is present in the most popular varieties of fluorescent proteins derived from Aequorea victoria, but is not the only mutation that improves folding at 37 degrees as other variants have been discovered.

In addition to improving the maturation at 37 degrees, the optimization of codon usage for mammalian expression has also improved overall brightness of green fluorescent protein expressed in mammalian cells. In all, over 190 silent mutations have been introduced into the coding sequence to enhance expression in human tissues. A Kozak translation initiation site (containing the nucleotide sequence A/GCCAT) was also introduced by insertion of valine as the second amino acid. These, along with a variety of other improvements (discussed below), have resulted in a very useful probe for live cell imaging of mammalian cells and are common to all of the currently used fluorescent probes derived from the original jellyfish protein.

The Fluorescent Protein Color Palette

A broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum (see Table 1). Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, and deep red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.
Fluorescent Protein Properties
Protein
(Acronym) Excitation
Maximum
(nm) Emission
Maximum
(nm) Molar
Extinction
Coefficient Quantum
Yield in vivo
Structure Relative
Brightness
(% of EGFP)
GFP (wt) 395/475 509 21,000 0.77 Monomer 48
Green Fluorescent Proteins
EGFP 484 510 56,000 0.60 Monomer 100
Emerald 487 509 57,500 0.68 Monomer 116
Azami Green 492 505 55,000 0.74 Monomer 121
CopGFP 482 502 70,000 0.60 Monomer 125
AcGFP 480 505 50,000 0.55 Monomer 82
ZsGreen 493 505 43,000 0.91 Tetramer 117
Blue Fluorescent Proteins
EBFP 383 445 29,000 0.31 Monomer 27
Sapphire 399 511 29,000 0.64 Monomer 55
T-Sapphire 399 511 44,000 0.60 Monomer 79
Cyan Fluorescent Proteins
AmCyan1 458 489 44,000 0.24 Tetramer 31
ECFP 439 476 32,500 0.40 Monomer 39
Cerulean 433 475 43,000 0.62 Monomer 79
Midoriishi Cyan 472 495 27,300 0.90 Dimer 73
Yellow Fluorescent Proteins
EYFP 514 527 83,400 0.61 Monomer 151
PhiYFP 525 537 130,000 0.40 Monomer 155
Citrine 516 529 77,000 0.76 Monomer 174
Venus 515 528 92,200 0.57 Monomer 156
ZsYellow1 529 539 20,200 0.42 Tetramer 25
Orange and Red Fluorescent Proteins
Kusabira Orange 548 559 51,600 0.60 Monomer 92
mOrange 548 562 71,000 0.69 Monomer 146
DsRed 558 583 75,000 0.79 Tetramer 176
DsRed2 563 582 43,800 0.55 Tetramer 72
DsRed-Express 555 584 38,000 0.51 Tetramer 58
mTangerine 568 585 38,000 0.30 Monomer 34
mStrawberry 574 596 90,000 0.29 Monomer 78
AsRed2 576 592 56,200 0.05 Tetramer 8
mRFP1 584 607 50,000 0.25 Monomer 37
mCherry 587 610 72,000 0.22 Monomer 47
HcRed1 588 618 20,000 0.015 Dimer 1
mRaspberry 598 625 86,000 0.15 Monomer 38
HcRed-Tandem 590 637 160,000 0.04 Monomer 19
mPlum 590 649 41,000 0.10 Monomer 12


Table 1

Presented in Table 1 is a compilation of properties displayed by several of the most popular and useful fluorescent protein variants. Along with the common name and/or acronym for each fluorescent protein, the peak absorption and emission wavelengths (given in nanometers), molar extinction coefficient, quantum yield, relative brightness, and in vivo structural associations are listed. The computed brightness values were derived from the product of the molar extinction coefficient and quantum yield, divided by the value for EGFP. This listing was created from scientific and commercial literature resources and is not intended to be comprehensive, but instead represents fluorescent protein derivatives that have received considerable attention in the literature and may prove valuable in research efforts. Furthermore, the absorption and fluorescence emission spectra listed in tables and illustrated below were recorded under controlled conditions and are normalized for comparison and display purposes only. In actual fluorescence microscopy investigations, spectral profiles and wavelength maxima may vary due to environmental effects, such as pH, ionic concentration, and solvent polarity, as well as fluctuations in localized probe concentration. Therefore, the listed extinction coefficients and quantum yields may differ from those actually observed under experimental conditions.

Green Fluorescent Proteins

Although native green fluorescent protein produces significant fluorescence and is extremely stable, the excitation maximum is close to the ultraviolet range. Because ultraviolet light requires special optical considerations and can damage living cells, it is generally not well suited for live cell imaging with optical microscopy. Fortunately, the excitation maximum of green fluorescent protein is readily shifted to 488 nanometers (in the cyan region) by introducing a single point mutation altering the serine at position 65 into a threonine residue (S65T). This mutation is featured in the most popular variant of green fluorescent protein, termed enhanced GFP (EGFP), which is commercially available in a wide range of vectors offered by BD Biosciences Clontech, one of the leaders in fluorescent protein technology. Furthermore, the enhanced version can be imaged using commonly available filter sets designed for fluorescein and is among the brightest of the currently available fluorescent proteins. These features have rendered enhanced green fluorescent protein one of the most popular probes and the best choice for most single-label fluorescent protein experiments. The only drawbacks to the use of EGFP are a slight sensitivity to pH and a weak tendency to dimerize.

In addition to enhanced green fluorescent protein, several other variants are currently being used in live-cell imaging. One of the best of these in terms of photostability and brightness may be the Emerald variant, but lack of a commercial source has limited its use. Several sources provide humanized green fluorescent protein variants that offer distinct advantages for fluorescence resonance energy transfer (FRET) experiments. Substitution of the phenylalanine residue at position 64 for leucine (F64L; GFP2) yields a mutant that retains the 400-nanometer excitation peak and can be coupled as an effective partner for enhanced yellow fluorescent protein. A variant of the S65C mutation (normally substituting cysteine for serine) having a peak excitation at 474 nanometers has been introduced commercially as a more suitable FRET partner for enhanced blue fluorescent protein than the red-shifted enhanced green version. Finally, a reef coral protein, termed ZsGreen1 and having an emission peak at 505 nanometers, has been introduced as a substitute for enhanced green fluorescent protein. When expressed in mammalian cells, ZsGreen1 is very bright relative to EGFP, but has limited utility in producing fusion mutants and, similar to other reef coral proteins, has a tendency to form tetramers.

Yellow Fluorescent Proteins

The family of yellow fluorescent proteins was initiated after the crystal structure of green fluorescent protein revealed that threonine residue 203 (Thr203) was near the chromophore. Mutation of this residue to tyrosine was introduced to stabilize the excited state dipole moment of the chromophore and resulted in a 20-nanometer shift to longer wavelengths for both the excitation and emission spectra. Further refinements led to the development of the enhanced yellow fluorescent protein (EYFP), which is one of the brightest and most widely used fluorescent proteins. The brightness and fluorescence emission spectrum of enhanced yellow fluorescent protein combine to make this probe an excellent candidate for multicolor imaging experiments in fluorescence microscopy. Enhanced yellow fluorescent protein is also useful for energy transfer experiments when paired with enhanced cyan fluorescent protein (ECFP) or GFP2. However, yellow fluorescent protein presents some problems in that it is very sensitive to acidic pH and loses approximately 50 percent of its fluorescence at pH 6.5. In addition, EYFP has also been demonstrated to be sensitive to chloride ions and photobleaches much more readily than the green fluorescent proteins.

Continued development of fluorescent protein architecture for yellow emission has solved several of the problems with the yellow probes. The Citrine variant of yellow fluorescent protein is very bright relative to EYFP and has been demonstrated to be much more resistant to photobleaching, acidic pH, and other environmental effects. Another derivative, named Venus, is the fastest maturing and one of the brightest yellow variants developed to date. The coral reef protein, ZsYellow1, originally cloned from a Zoanthus species native to the Indian and Pacific oceans, produces true yellow emission and is ideal for multicolor applications. Like ZsGreen1, this derivative is not as useful for creating fusions as EYFP and has a tendency to form tetramers. Many of the more robust yellow fluorescent protein variants have been important for quantitative results in FRET studies and are potentially useful for other investigations as well.

Illustrated in Figure 3 are the absorption and emission spectral profiles for many of the commonly used and commercially available fluorescent proteins, which span the visible spectrum from cyan to far red. The variants derived from Aequorea victoria jellyfish, including enhanced cyan, green, and yellow fluorescent proteins, exhibit peak emission wavelengths ranging from 425 to 525 nanometers. Fluorescent proteins derived from coral reefs, DsRed2 and HcRed1 (discussed below), emit longer wavelengths but suffer from oligomerization artifacts in mammalian cells.

Blue and Cyan Fluorescent Proteins

The blue and cyan variants of green fluorescent protein resulted from direct modification of the tyrosine residue at position 66 (Tyr66) in the native fluorophore (see Figure 2). Conversion of this amino acid to histidine results in blue emission having a wavelength maxima at 450 nanometers, whereas conversion to tryptamine results in a major fluorescence peak around 480 nanometers along with a shoulder that peaks around 500 nanometers. Both probes are only weakly fluorescent and require secondary mutations to increase folding efficiency and overall brightness. Even with modifications, the enhanced versions in this class of fluorescent protein (EBFP and ECFP) are only about 25 to 40 percent as bright as enhanced green fluorescent protein. In addition, excitation of blue and cyan fluorescent proteins is most efficient in spectral regions that are not commonly used, so specialized filter sets and laser sources are required.

Despite the drawbacks of blue and cyan fluorescent proteins, the widespread interest in multicolor labeling and FRET has popularized their application in a number of investigations. This is especially true for enhanced cyan fluorescent protein, which can be excited off-peak by an argon-ion laser (using the 457-nanometer spectral line) and is significantly more resistant to photobleaching than the blue derivative. In contrast to other fluorescent proteins, there has not been a high level of interest for designing better probes in the blue region of the visible light spectrum, and a majority of the developmental research on fluorophores in this class has been focused on cyan variants.

Among the improved cyan fluorescent proteins that have been introduced, AmCyan1 and an enhanced cyan variant termed Cerulean show the most promise. Derived from the reef coral, Anemonia majano, the AmCyan1 fluorescent protein variant has been optimized with human codons to generate a high relative brightness level and resistance to photobleaching when compared to enhanced cyan fluorescent protein during mammalian expression. On the downside, similar to most of the other reef coral proteins, this probe has a tendency to form tetramers. The Cerulean fluorescent probe was developed by site-directed mutagenesis of enhanced cyan fluorescent protein to yield a higher extinction coefficient and improved quantum yield. Cerulean is at least 2-fold brighter than enhanced cyan fluorescent protein and has been demonstrated to significantly increase the signal-to-noise ratio when coupled with yellow-emitting fluorescent proteins, such as Venus (see Figure 4), in FRET investigations.

Red Fluorescent Proteins

A major goal of fluorescent protein development has become the construction of a red-emitting derivative that equals or exceeds the advanced properties of enhanced green fluorescent protein. Among the advantages of a suitable red fluorescent protein are the potential compatibility with existing confocal and widefield microscopes (and their filter sets), along with an increased capacity to image entire animals, which are significantly more transparent to red light. Because the construction of red-shifted mutants from the Aequorea victoria jellyfish green fluorescent protein beyond the yellow spectral region has proven largely unsuccessful, investigators have turned their search to the tropical reef corals.

The first coral-derived fluorescent protein to be extensively utilized was derived from Discosoma striata and is commonly referred to as DsRed. Once fully matured, the fluorescence emission spectrum of DsRed features a peak at 583 nanometers whereas the excitation spectrum has a major peak at 558 nanometers and a minor peak around 500 nanometers. Several problems are associated with using DsRed, however. Maturation of DsRed fluorescence occurs slowly and proceeds through a time period when fluorescence emission is in the green region. Termed the green state, this artifact has proven problematic for multiple labeling experiments with other green fluorescent proteins because of the spectral overlap. Furthermore, DsRed is an obligate tetramer and can form large protein aggregates in living cells. Although these features are inconsequential for the use of DsRed as a reporter of gene expression, the usefulness of DsRed as an epitope tag is severely limited. In contrast to the jellyfish fluorescent proteins, which have been successfully used to tag hundreds of proteins, DsRed conjugates have proven much less successful and are often toxic.

A few of the problems with DsRed fluorescent proteins have been overcome through mutagenesis. The second-generation DsRed, known as DsRed2, contains several mutations at the peptide amino terminus that prevent formation of protein aggregates and reduce toxicity. In addition, the fluorophore maturation time is reduced with these modifications. The DsRed2 protein still forms a tetramer, but it is more compatible with green fluorescent proteins in multiple labeling experiments due to the quicker maturation. Further reductions in maturation time have been realized with the third generation of DsRed mutants, which also display an increased brightness level in terms of peak cellular fluorescence. Red fluorescence emission from DsRed-Express can be observed within an hour after expression, as compared to approximately six hours for DsRed2 and 11 hours for DsRed. A yeast-optimized variant, termed RedStar, has been developed that also has an improved maturation rate and increased brightness. The presence of a green state in DsRed-Express and RedStar is not apparent, rendering these fluorescent proteins the best choice in the orange-red spectral region for multiple labeling experiments. Because these probes remain obligate tetramers, they are not the best choice for labeling proteins.

Several additional red fluorescent proteins showing a considerable amount of promise have been isolated from the reef coral organisms. One of the first to be adapted for mammalian applications is HcRed1, which was isolated from Heteractis crispa and is now commercially available. HcRed1 was originally derived from a non-fluorescent chromoprotein that absorbs red light through mutagenesis to produce a weakly fluorescent obligate dimer having an absorption maximum at 588 nanometers and an emission maximum of 618 nanometers. Although the fluorescence emission spectrum of this protein is adequate for separation from DsRed, it tends to co-aggregate with DsRed and is far less bright. An interesting HcRed construct containing two molecules in tandem has been produced to overcome dimerization that, in principle, occurs preferentially within the tandem pairing to produce a monomeric tag. However, because the overall brightness of this twin protein has not yet been improved, it is not a good choice for routine applications in live-cell microscopy.

Developing Monomeric Fluorescent Protein Variants

In their natural states, most fluorescent proteins exist as dimers, tetramers, or higher order oligomers. Likewise, Aequorea victoria green fluorescent protein is thought to participate in a tetrameric complex with aequorin, but this phenomenon has only been observed at very high protein concentrations and the tendency of jellyfish fluorescent proteins to dimerize is generally very weak (having a dissociation constant greater than 100 micromolar). Dimerization of fluorescent proteins has thus not generally been observed when they are expressed in mammalian systems. However, when fluorescent proteins are targeted to specific cellular compartments, such as the plasma membrane, the localized protein concentration can theoretically become high enough to permit dimerization. This is a particular concern when conducting FRET experiments, which can yield complex data sets that are easily compromised by dimerization artifacts.

The construction of monomeric DsRed variants has proven to be a difficult task. More than 30 amino acid alterations to the structure were required for the creation of the first-generation monomeric DsRed protein (termed RFP1). However, this derivative exhibits significantly reduced fluorescence emission compared to the native protein and photobleaches very quickly, rendering it much less useful then monomeric green and yellow fluorescent proteins. Mutagenesis research efforts, including novel techniques such as somatic hypermutation, are continuing in the search for yellow, orange, red, and deep red fluorescent protein variants that further reduce the tendency of these potentially efficacious biological probes to self-associate while simultaneously pushing emission maxima towards longer wavelengths.

Improved monomeric fluorescent proteins are being developed that have increased extinction coefficients, quantum yields, and photostability, although no single variant has yet been optimized by all criteria. In addition, the expression problems with obligate tetrameric red fluorescent proteins are being overcome by the efforts to generate monomeric variants, which have yielded derivatives that are more compatible with biological function.

Perhaps the most spectacular development on this front has been the introduction of a new harvest of fluorescent proteins derived from monomeric red fluorescent protein through directed mutagenesis targeting the Q66 and Y67 residues. Named for fruits that reflect colors similar to the fluorescence emission spectral profile (see Table 1 and Figure 5), this cadre of monomeric fluorescent proteins exhibits maxima at wavelengths ranging from 560 to 610 nanometers. Further extension of this class through iterative somatic hypermutation yielded fluorescent proteins with emission wavelengths up to 650 nanometers. These new proteins essentially fill the gap between the most red-shifted jellyfish fluorescent proteins (such as Venus), and the coral reef red fluorescent proteins. Although several of these new fluorescent proteins lack the brightness and stability necessary for many imaging experiments, their existence is encouraging as it suggests the eventuality of bright, stable, monomeric fluorescent proteins across the entire visible spectrum.

Optical Highlighters

One of the most interesting developments in fluorescent protein research has been the application of these probes as molecular or optical highlighters (see Table 2), which change color or emission intensity as the result of external photon stimulation or the passage of time. As an example, a single point mutation to the native jellyfish peptide creates a photoactivatable version of green fluorescent protein (known as PA-GFP) that enables photoconversion of the excitation peak from ultraviolet to blue by illumination with light in the 400-nanometer range. Unconverted PA-GFP has an excitation peak similar in profile to that of the wild type protein (approximately 395 to 400 nanometers). After photoconversion, the excitation peak at 488 nanometers increases approximately 100-fold. This event evokes very high contrast differences between the unconverted and converted pools of PA-GFP and is useful for tracking the dynamics of molecular subpopulations within a cell. Illustrated in Figure 6(a) is a transfected living mammalian cell containing PA-GFP in the cytoplasm being imaged with 488-nanometer argon-ion laser excitation before (Figure 6(a)) and after (Figure 6(d)) photoconversion with a 405-nanometer blue diode laser.
Properties of Optical Highlighters
Protein
(Acronym) Excitation
Maximum
(nm) Emission
Maximum
(nm) Molar
Extinction
Coefficient
Quantum
Yield in vivo
Structure Relative
Brightness
(% of EGFP)
PA-GFP 504 517 17,400 0.79 Monomer 41
CoralHue Kaede (G) 508 518 98,800 0.88 Tetramer 259
CoralHue Kaede (R) 572 580 60,400 0.33 Tetramer 59
CoralHue Dronpa (G) 503 518 95,000 0.85 Monomer 240
Kindling (KFP1) 580 600 59,000 0.07 Tetramer 12
PS-CFP (C) 402 468 34,000 0.16 Monomer 16
PS-CFP (G) 490 511 27,000 0.19 Monomer 15
mEosFP (G) 505 516 67,200 0.64 Monomer 128
mEosFP (O) 569 581 37,000 0.62 Monomer 68


Table 2

Other fluorescent proteins can also be employed as optical highlighters. Three-photon excitation (at less than 760 nanometers) of DsRed fluorescent protein is capable of converting the normally red fluorescence to green. This effect is likely due to selective photobleaching of the red chromophores in DsRed, resulting in observable fluorescence from the green state. The Timer variant of DsRed gradually turns from bright green (500-nanometer emission) to bright red (580-nanometer emission) over the course of several hours. The relative ratio of green to red fluorescence can then be used to gather temporal data for gene expression investigations.

A photoswitchable optical highlighter, termed PS-CFP, derived by mutagenesis of a green fluorescent protein variant, has been observed to transition from cyan to green fluorescence upon illumination at 405 nanometers (note photoconversion of the central cell in Figures 6(b) and 6(e)). Expressed as a monomer, this probe is potentially useful in photobleaching, photoconversion and photoactivation investigations. However, the fluorescence from PS-CFP is approximately 2.5-fold dimmer than PA-GFP and is inferior to other highlighters in terms of photoconversion efficiency (the 40-nanometer shift in fluorescence emission upon photoconversion is less than observed with similar probes). Additional mutagenesis of this or related fluorescent proteins has the potential to yield more useful variants in this wavelength region.

Optical highlighters have also been developed in fluorescent proteins cloned from coral and anemone species. Kaede, a fluorescent protein isolated from stony coral, photoconverts from green to red in the presence of ultraviolet light. Unlike PA-GFP, the conversion of fluorescence in Kaede occurs by absorption of light that is spectrally distinct from its illumination. Unfortunately, this protein is an obligate tetramer, making it less suitable fur use as an epitope tag than PA-GFP. Another tetrameric stony coral (Lobophyllia hemprichii) fluorescent protein variant, termed EosFP (see Table 2), emits bright green fluorescence that changes to orange-red when illuminated with ultraviolet light at approximately 390 nanometers. In this case, the spectral shift is produced by a photo-induced modification involving a break in the peptide backbone adjacent to the chromophore. Further mutagenesis of the "wild type" EosFP protein yielded monomeric derivatives, which may be useful in constructing fusion proteins.

A third non-Aequorea optical highlighter, the Kindling fluorescent protein (KFP1) has been developed from a non-fluorescent chromoprotein isolated in Anemonia sulcata, and is now commercially available (Evrogen). Kindling fluorescent protein does not exhibit emission until illuminated with green light. Low-intensity light results in a transient red fluorescence that decays over a few minutes (see the mitochondria in Figure 6(c)). Illumination with blue light quenches the kindled fluorescence immediately, allowing tight control over fluorescent labeling. In contrast, high-intensity illumination results in irreversible kindling and allows for stable highlighting similar to PA-GFP (Figure 6(f)). The ability to precisely control fluorescence is particular useful when tracking particle movement in a crowded environment. For example, this approach has been successfully used to track the fate of neural plate cells in developing Xenopus embryos and the movement of individual mitochondria in PC12 cells.

As the development of optical highlighters continues, fluorescent proteins useful for optical marking should evolve towards brighter, monomeric variants that can be easily photoconverted and display a wide spectrum of emission colors. Coupled with these advances, microscopes equipped to smoothly orchestrate between illumination modes for fluorescence observation and regional marking will become commonplace in cell biology laboratories. Ultimately, these innovations have the potential to make significant achievements in the spatial and temporal dynamics of signal transduction systems.

Fluorescent Protein Vectors and Gene Transfer

Fluorescent proteins are quite versatile and have been successfully employed in almost every biological discipline from microbiology to systems physiology. These ubiquitous probes have been extremely useful as reporters for gene expression studies in cultured cells and tissues, as well as living animals. In live cells, fluorescent proteins are most commonly employed to track the localization and dynamics of proteins, organelles, and other cellular compartments. A variety of techniques have been developed to construct fluorescent protein fusion products and enhance their expression in mammalian and other systems. The primary vehicles for introducing fluorescent protein chimeric gene sequences into cells are genetically engineered bacterial plasmids and viral vectors.

Fluorescent protein gene fusion products can be introduced into mammalian and other cells using the appropriate vector (usually a plasmid or virus) either transiently or stably. In transient, or temporary, gene transfer experiments (often referred to as transient transfection), plasmid or viral DNA introduced into the host organism does not necessarily integrate into the chromosomes, but can be expressed in the cytoplasm for a short period of time. Expression of gene fusion products, easily monitored by the observation of fluorescence emission using a filter set compatible with the fluorescent protein, usually takes place over a period of several hours after transfection and continues for 72 to 96 hours after introduction of plasmid DNA into mammalian cells. In many cases, the plasmid DNA can be incorporated into the genome in a permanent state to form stably transformed cell lines. The choice of transient or stable transfection depends upon the target objectives of the investigation.

The basic plasmid vector configuration useful in fluorescent protein gene transfer experiments has several requisite components. The plasmid must contain prokaryotic nucleotide sequences coding for a bacterial replication origin for DNA and an antibiotic resistance gene. These elements, often termed shuttle sequences, allow propagation and selection of the plasmid within a bacterial host to generate sufficient quantities of the vector for mammalian transfections. In addition, the plasmid must contain one or more eukaryotic genetic elements that control the initiation of messenger RNA transcription, a mammalian polyadenylation signal, an intron (optional), and a gene for co-selection in mammalian cells. Transcription elements are necessary for the mammalian host to express the gene fusion product of interest, and the selection gene is usually an antibiotic that bestows resistance to cells containing the plasmid. These general features vary according to plasmid design, and many vectors have a wide spectrum of additional components suited for particular applications.

Illustrated in Figure 7 is the restriction enzyme and genetic map of a commercially available (BD Biosciences Clontech) bacterial plasmid derivative containing the coding sequence for enhanced yellow fluorescent protein fused to the endoplasmic reticulum targeting sequence of calreticulin (a resident protein). Expression of this gene product in susceptible mammalian cells yields a chimeric peptide containing EYFP localized to the endoplasmic reticulum membrane network, designed specifically for fluorescent labeling of this organelle. The host vector is a derivative of the pUC high copy number (approximately 500) plasmid containing the bacterial replication origin, which makes it suitable for reproduction in specialized E. coli strains. The kanamycin antibiotic gene is readily expressed in bacteria and confers resistance to serve as a selectable marker.

Additional features of the EYFP vector presented above are a human cytomegalovirus (CMV) promoter to drive gene expression in transfected human and other mammalian cell lines, and an f1 bacteriophage replication origin for single-stranded DNA production. The vector backbone also contains a simian virus 40 (SV40) replication origin, which is active in mammalian cells that express the SV40 T-antigen. Selection of stable transfectants with the antibiotic G418 is enabled with a neomycin resistance cassette consisting of the SV40 early promoter, the neomycin resistance gene (aminoglycoside 3’-phosphotransferase), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV-TK) for messenger stability. Six unique restriction enzyme sites (see Figure 7) are present on the plasmid backbone, which increases the versatility of this plasmid.

Propagation, Isolation, and Transfection of Fluorescent Protein Plasmids

Successful mammalian transfection experiments rely on the use of high quality plasmid or viral DNA vectors that are relatively free of bacterial endotoxins. In the native state, circular plasmid DNA molecules exhibit a tertiary supercoiled conformation that twists the double helix around itself several times. For many years, the method of choice for supercoiled plasmid and virus DNA purification was cesium chloride density gradient centrifugation in the presence of an intercalation agent (such as ethidium bromide or propidium iodide). This technique, which is expensive in terms of both equipment and materials, segregates the supercoiled (plasmid) DNA from linear chromosomal and nicked circular DNA according to buoyant density, enabling the collection of high purity plasmid DNA. Recently, simplified ion-exchange column chromatography methods (commonly termed a mini-prep) have largely supplanted the cumbersome and time-consuming centrifugation protocol to yield large quantities of endotoxin-free plasmid DNA in a relatively short period of time.

Specialized bacterial mutants, termed competent cells, have been developed for convenient and relatively cheap amplification of plasmid vectors. The bacteria contain a palette of mutations that render them particularly susceptible to plasmid replication, and have been chemically permeabilized for transfer of the DNA across the membrane and cell wall in a procedure known as transformation. After transformation, the bacteria are grown to logarithmic phase in the presence of the selection antibiotic dictated by the plasmid. The bacterial culture is concentrated by centrifugation and disrupted by lysis with an alkaline detergent solution containing enzymes to degrade contaminating RNA. The lysate is then filtered and placed on the ion-exchange column. Unwanted materials, including RNA, DNA, and proteins, are thoroughly washed from the column before the plasmid DNA is eluted using a high salt buffer. Alcohol (isopropanol) precipitation concentrates the eluted plasmid DNA, which is collected by centrifugation, washed, and redissolved in buffer. The purified plasmid DNA is ready for duty in transfection experiments.

Mammalian cells used for transfection must be in excellent physiological condition and growing in logarithmic phase during the procedure. A wide spectrum of transfection reagents has been commercially developed to optimize uptake of plasmid DNA by cultured cells. These techniques range from simple calcium phosphate precipitation to sequestering the plasmid DNA in lipid vesicles that fuse to the cell membrane and deliver the contents to the cytoplasm (as illustrated in Figure 8). Collectively termed lipofection, the lipid-based technology has met with widespread acceptance due to its effectiveness in a large number of popular cell lines, and it is now the method of choice for most transfection experiments.

Although transient transfections usually result in the loss of plasmid gene product over a relatively short period of time (several days), stably transfected cell lines continue to produce the guest proteins on a continuous long-term basis (ranging from months to years). Stable cell lines can be selected using antibiotic markers present in the plasmid backbone (see Figure 7). One of the most popular antibiotics for selection of stable transfectants in mammalian cell lines is the protein synthesis-inhibiting drug G418, but the required dose varies widely according to each cell line. Other common antibiotics, including hydromycin-B and puromycin, have also been developed for stable cell selection, as have genetic markers. The most efficient method of obtaining stable cell lines is to employ a high efficiency technique for the initial transfection. In this regard, electroporation has proven to generate stable transfectants with linearized plasmids and purified genes. Electroporation applies short, high voltage pulses to a cellular suspension to induce pore formation in the plasma membrane, subsequently allowing the transfection DNA to enter the cell. Specialized equipment is necessary for electroporation, however, the technique is comparable in expense to lipofection reagents when a large number of transfections are performed.

The Future of Fluorescent Proteins

The focus of current fluorescent protein development is centered on two basic goals. The first is to perfect and fine-tune the current palette of blue to yellow fluorescent proteins derived from Aequorea victoria jellyfish, while the second aim is to develop monomeric fluorescent proteins emitting in the orange to far red regions of the visible light spectrum. Progress toward these goals has been quite impressive, and it is not inconceivable that near-infrared fluorescent proteins loom on the horizon.

The latest generation of jellyfish variants has solved most of the deficiencies of the first generation fluorescent proteins, particularly for the yellow and green derivatives. The search for a monomeric, bright, and fast-maturing red fluorescent protein has resulted in several new and interesting classes of fluorescent proteins, particularly those derived from coral species. Development of existing fluorescent proteins, together with new technologies, such as insertion of unnatural amino acids, will further expand the color palette. As optical spectral separation techniques become better developed and more widespread, these new varieties will supplement the existing palette, especially in the yellow and red regions of the spectrum.

The current trend in fluorescent probe technology is to expand the role of dyes that fluoresce into the far red and near infrared. In mammalian cells, both autofluorescence and the absorption of light are greatly reduced at the red end of the spectrum. Thus, the development of far red fluorescent probes would be extremely useful for the examination of thick specimens and entire animals. Given the success of fluorescent proteins as reporters in transgenic systems, the use of far red fluorescent proteins in whole organisms will become increasingly important in the coming years.

Finally, the tremendous potential in fluorescent protein applications for the engineering of biosensors is just now being realized. The number of biosensor constructs is rapidly growing. By using structural information, development of these probes has led to improved sensitivity and will continue to do so. The success of these endeavors certainly suggests that almost any biological parameter will be measurable using the appropriate fluorescent protein-based biosensor.

BACK TO FLUORESCENCE MICROSCOPY

Contributing Authors

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, 37232.

George H. Patterson and Jennifer Lippincott-Schwartz - Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892.
Nathan S. Claxton and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

GFP, RFP

This web page was produced as an assignment for an undergraduate course at Davidson College
Red Fluorescent Protein

Figure 1. Image of DsRed (red fluorescent protein) and
and green fluorescent protein (GFP). Image used with
permission from Clontech.

Reporter genes are valuable tools in molecular biology because they allow researchers to visualize the protein products of genes. Green fluorescent protein (GFP) is unique because it naturally fluoresces with out the addition of substrates or enzymes, allowing researchers to view genetic expression in living cells. GFP can serve as a transcription marker or it can be used to visualize protein localization. The newly discovered red fluorescent protein (RFP) shares many properties of GFP and can be applied in a similar manner. One form of red fluorescent protein (DsRed), isolated from Discosoma striata and manufactured by Clontech, has an optimal absorption at 558 nm and emits light at 583 nm (CLONTECH, 1999). Another form of RFP (cob A) has been isolated from Propionibacterium freudenreichii (Wildt and Deuschle, 1999). One advantage of red fluorescent protein (RFP) is that it produces less background interference than GFP. Although the greatest advantage of RFP is that it can be used in conjunction with GFP to colabel cells.

Expression System

Cloning and Fusion
RFP is a reporter gene that allows the visual detection of gene expression in living cells. This is how it works. First the RFP gene must be fused to a gene of interest. A commercially bought RFP gene is already inserted into a plasmid (there are various types of plasmids available that have been optimized for different types of research). RFP gene plasmids (pRFP) have polylinkers located near the RFP gene. The gene of interest and pRFP are cut with a restriction enzyme and mixed together so they can ligate. Once the gene of interest is ligated to the RFP plasmid, it can be cloned by inserting the pRFP into bacterial cells. Depending on the plasmid being used, an ampR gene or another gene conferring resistance may also be found on pRFP. Thus transformed bacteria can be selected by their resistance to a substance. Again depending on the type of pRFP purchased, a promoter many need to be inserted into the plasmid or the plasmid may already contain a promoter. The plasmid can now be cloned via bacterial replication.


Expression
The vectors created in the previous steps can be inserted into any living cells. Once inside, the chimeric protein will be expressed through the natural process of transcription and translation. This protein should contain a functional RFP and protein of interest. A light of the appropriate wavelength must be shown on the cells and the RFP will fluoresce, allowing visual detection of the protein of interest.


Plasmids available from Clontech

Figure 2. Plasmids containing the RFP gene that are currently available from Clontech.
Image used with permission from Clontech.

pDsRed: This is a cDNA copy of the RFP gene (DsRed). It is flanked by two polylinkers (MCS-multiple cloning sites). This plasmid also contains ampR and the lac promoter. Thus the RFP gene can be removed and inserted into a new plasmid or this plasmid can be inserted into the appropriate vector. This plasmid is optimal for nonmammalian expression.

pDsRed1-1: This plasmid contains an RFP gene that has been altered for human expression of RFP (DsRed1). A promoter must be inserted into this plasmid for RFP to be expressed. Rather than create a fusion protein, this vector is ideal for research on promoter and enhancer sequences in mammals.

pDsRed1-N1: This plasmid also contains DsRed1 as well as a CMV promoter, making it ideal for creating fusion proteins to be expressed in mammalian cells.


Microscopy
The expression of RFP can be visualized using a fluorescence microscope with a rhodamine or FITC filter set (Wildt and Deuschle, 1999). Colabelling is possible by transfecting cells with different plasmids each containing one fluorescent protein gene and one gene of interest (Figure 3). Double or even triple labeling a cell is a highly informative tool. Now researchers can see the interaction between gene products or the difference in localization between two or more protiens. Another possible experiment is to monitor the progressional expression of two or more genes, which would be very useful in developmental studies.


Figure 3. HeLa Cell transfected with three fusion fluorescent
proteins. Nuclear proteins fused with enhanced cyan fluorescent
protein (ECFP). Tubulin proteins labeled with EY(yellow) FP.
Mitochondrial proteins were labeled with DsRed. Image used with
permission from Clontech.


Flow Cytometry
Flow cytometry allows a mixed sample of cells to be sorted their physical or chemical properties. Cells that are unusual in some way can be collected for further research. Cells are labeled with a fluorescent dye that can be excited by a beam of light. Cells that emit fluorescence are detected and this information is stored for further study. In some flow cytometry devices fluorescing cells are sequestered. DsRed serves as such a dye. A 488nm argon laser can be used to excite DsRed in flow cytometry experiments (CLONTECH, 1999).

All questions concerning the purchase of this product should be directed to Clontech
Works Consulted


Campbell, N. A. 1996. Biology, 4th ed. Menlo Park, CA: Benjamin/Cummings Publishing Company, Inc., . p. 372-374.

CLONTECH Laboratories. 1999. Living Colors Red Fluorescent Protein. CLONTECHniques.October:2-6.
accessed 2000 13 Feb.

The John Curtin School of Medical Research. 1998 March 12. JCSMR Flow Cytometry "An introduction".
.Accessed 2000 20 Feb.

Wildt, S and Deuschle, U. 1999. cobA, a red fluorescent transcriptional reporter for Escherichia
coli, yeast, and mammalian cells. Nature Biotechnology. 17: 1175-1178.
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