F2 and F1 screening
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This article lists the steps involved in F2 and F1 screens, and compares and contrasts the two techniques.
F2 screen
Wild-type (wt) males treated with chemical mutagen (such as ENU, dependant on the type of mutation desired) to generate mutant sperm and crossed with untreated wild-type females.
Each of the F1 offspring will be heterozygous for a unique mutation. This is because each sperm is haploid (n), and each mutation is statistically likely to occur in a different place on the genome.
F1 STAGE Offspring individually mated with wild-type to generate F2 families/lines.
The F2 offspring will have a 1:1 chance of being either wt homozygous or heterozygous wt/mutant (mut).
F2 STAGE The F2 generation are mated randomly and individually with each other. 25% of the crosses will be heterozygous x heterozygous.
Of the lines created from above, the line of interest is the heterozygous x heterozygous line, as this line alone gives the possibility of a homozygous mutant offspring. As the majority of mutations are recessive, they will be visible in homozygous mutant offspring.
F3 STAGE From the 3 possible combinations of crosses of the F2 offspring, the F3 lines will have either normal phenotypes (0.75 probability) or mutant phenotypes (0.25 probability). Only the ‘mutant’ line will be of interest, in which ~25% of the individuals will display the mutant characteristics (most of the time a homozygous mutation is lethal).
-->The parents of the mutant lines can be kept for further analysis and characteristics of the mutant type.
F1 screen
Wild-type (wt) males treated with chemical mutagen (such as ENU, dependant on the type of mutation desired) to generate mutant sperm and crossed with untreated wild-type females. Each of the F1 offspring will be heterozygous for a unique mutation. This is because each sperm is haploid (n), and each mutation is statistically likely to occur in a different place on the genome.
F1 STAGE Heterozygous females eggs are mixed with irradiated sperm. [NB This is almost the same step as the F2 cross F1 stage] Sperm are irradiated using UV light; in this way the cytosols of the sperm and egg can mix and trigger normal development of the embryo for 24 hours, without any of the genetic information of the sperm.
F2 STAGE Embryos of the F1 cross can be screened for developmental defects in the first 24 hours. Note although half of the embryos of the F2 will have mutations, not all of these will affect development in the first 24 hours.
F1 notes and generating a stable mutant stock from F1 screen embryos:
It should be noted that to date, the F1 screen is only possible in Zebrafish, this is due to a curious property of Zebrafish haploid embryos to continue development for a limited time (24 hours). As a consequence of this interesting mutations cannot be maintained from the embryo, as it is haploid and will not develop into a fertile adult. To maintain a stock of fish carrying this mutation, the adult heterozygous female ‘mother’ of the haploid embryo of interest can be bred in the same way as the F2 screen, from step F1.
In order to keep a line of mutants identified using either screen, sperm of males from the F2 line which produces mutant offspring (i.e. heterozygous mutant sperm) can be cryogenically frozen, to be revived as and when needed.
F1 vs F2 screens
Screening programs are necessary to identify new mutations for analysis, as spontaneous mutations are rare, and when they do occur they are often not visible. This is because mutations occur on only one chromosome and so are ‘masked’ by the wild-type gene on the other chromosome – i.e. they are phenotypically wild-type. Spontaneous mutations are rare, occurring in Zebrafish for example in only 1 in 10 000 embryos. Inducing mutations using a mutagen can increase this rate to about 1 in 300 gametes. The site at which mutations occur is relatively random, and each mutation is likely to be unique, although two mutations may occur at two different loci on the same chromosome. Thus F2 screens are a dependable method of generating mutant lines, and it is almost a guarantee that from a project starting with only a single fish, c. 300 different mutants can be found. However F2 screening for new mutations involves raising vast numbers of the organisms used for screening, which using Zebrafish as an example can involve several thousand fish. Each generation of zebrafish requires 3 to 4 months to reach maturity, in which case around a year of breeding is required to identify the mutants in the F3 generation. Individual mating required of the F1 and F2 stage crosses (in the F2 program) can be very labour intensive, and separate holding facilities for each line are required. In Zebrafish, with a brood size of around 300, around 300 separate tanks would be required for the F1 generation crosses alone. In addition to the space and labour time required the organisms must be fed. All of this inevitably requires a substantial amount of funding. An F1 screen has the obvious advantage that it involves a generation less than the F2 screen, using about only a tenth of the number of fish that an F2 screen uses. However haploid emryos can often develop abnormally, and so many false positives occur, where a defect is thought to be as a result of a new mutation but maybe a result of haploid development. Of course with an F1 screen it is still necessary to use an F2 breeding program to generate stable lines of fish for analysis, which has some of the disadvantages of said program inherent. A double edged sword of the F1 screen is that it can only be used to identify developmental defects occurring within the first 24 hours of fertilisation. This is useful as it allows developmental defects to be examined more easily and for less funding but means many developmental and other defects can be easily missed.
See also
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