Basic Methods of Culturing Drosophila（培养果蝇基本方法）
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Stockkeeping
1.
Mechanics
Most stocks can be successfully cultured by periodic mass transfer of adults to fresh food. Bottles or vials are tapped on the pounding pad to shake flies away from the plug, the plug is rapidly removed and the old culture inverted over a fresh bottle or vial. Flies are tapped into the new vessel, or some shaken back into the old one, as necessary, and the two are rapidly separated and replugged. Good tossing technique combined with plugs that are easily removed and replaced result in very few escapees. You will learn from experience which stocks require a medium or large inoculation of adults and which do better with only a few.
The frequency with which new subcultures need to be established depends on the health and fecundity of the genotype, the temperature at which it is raised, and the density of the cultures. Temperature has a large effect on the rate of Drosophila development. Generation time (from egg to adult) is approximately: 7 days at 29°C, 9 days at 25°C, 11 days at 22°C, 19 days at 18°C. For most purposes stocks are maintained by live culture, transferring adults to fresh medium every few generations. Stocks kept at room temperature should be transferred to fresh food every 20 to 30 days. Mites and mold are more likely to be a problem in older stocks, so it is good policy to set 30 days as an absolute upper limit for room temperature stocks. This period can be extended by keeping stocks at lower temperature. 18°C buys more time than 22°C, for example, but a significant number of genotypes fail to thrive at 18°C, and mold can be a serious problem. It is wise to keep a room temperature backup of stocks to be maintained at low temperature for the first two or three transfers in case the stocks do poorly. If the quality of your fly food is unreliable it is wise to have at least two cultures for each stock, staggered to assure the use of different batches of medium (at least until you find a new cook).
Cryopreservation of ovaries (see Ashburner, 1989 ) or embryos ( Cole et al., 1993 ; Steponkus and Caldwell, 1993 ) are viable alternatives to continuous culture for some purposes. Genotypes that are unstable due to reversion, breakdown, or accumulation of modifiers, especially those with non-visible phenotypes that are time consuming to select, are good candidates for freezing. Also, if you are generating hundreds of stocks that will not be in use but must be kept for many years it might be cost-effective to maintain these as frozen stocks. For most routine stockkeeping purposes, however, live culture remains the preferred route.
Identify stocks with tags showing the complete genotype of the stock, sans shorthand. Writing the genotype on the vial or bottle at each transfer invites transcription errors and takes longer than moving a tag. Don"t use a stock center stock number or other potentially cryptic symbol as the only identifier of a stock. Stocks are often kept for many years and what is obvious to you now may be meaningless even to you in a few years, and is easily misinterpreted by someone inheriting your stocks. Unless you are careful to maintain complete stock data elsewhere, record all relevant information on the tag.
2.
Balanced stocks and balancers
Mutations that are homozygous viable and fertile are most easily kept as homozygous stocks. Lethal or sterile mutations must be maintained in a heterozygous state. A balanced stock is one that regenerates the same set of heterozygotes each generation so the stock can be maintained by mass transfer of adults instead of by mating specific genotypes each generation. A simple balanced lethal stock carries different recessive lethals on each of the two homologues, allowing only heterozygotes to survive. Dominant male or female steriles (Ms or Fs) can be maintained in stock without selection by double balancing - one Ms, one Fs and one recessive lethal. Fs/lethal male and Ms/lethal females will be the only fertile genotypes present in the stock each generation.
In most cases, balanced lethal schemes work only if one of the lethal chromosomes is itself a balancer chromosome. Recombination between lethal (or sterile) mutations on different homologues can produce one homologue with both mutations and one wild-type homologue. The wild-type chromosome will rapidly predominate or become fixed in the stock. Balancers are structurally rearranged chromosomes that prevent recombination between homologues in females (meiotic recombination is absent in D. melanogaster males and in the tiny 4th chromosome in females). This is accomplished in part by reducing recombination directly and in part by preventing transmission of recombinant chromosomes. The most commonly used balancers carry overlapping sets of inversions and prevent recombination throughout most of the length of the chromosome. Some special purpose balancers work well only for specific regions of a chromosome. Suppression of recombination is less effective when balancers for two or more heterologues are present in a stock.
