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Comment
. 2013 Feb;193(2):377-81.
doi: 10.1534/genetics.112.148379.

Targeted gene replacement in Drosophila goes the distance

K Nicole Crown, Jeff Sekelsky
Comment

Targeted gene replacement in Drosophila goes the distance

K Nicole Crown et al. Genetics. 2013 Feb.
No abstract available

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Figures

Figure 1
Figure 1
The steps typically taken to achieve gene replacement are listed. Many factors can affect the total time to complete each procedure, but readers with a basic knowledge of Drosophila genetics may be able to estimate the minimum time for each.
Figure 2
Figure 2
Long-range SIRT. In step 1, a recombineered vector (pP[Walkman]) containing the modified region of interest, an attB site (53 bp), an I-SceI recognition site (18 bp), two FRT sites (34 bp), and two P-element ends (100–500 bp) is injected into stocks that contain an attP site within ~70 kb of the region of interest. Recombination between attP and attB results in attL and attR sites flanking the insertion. In step 2, a double-strand break is induced at the I-SceI site, and repair of that break results in collapse of the duplication. Either the modified or the endogenous region of interest can be retained. The pP[Walkman] backbone can be removed through a FLP/FRT recombination reaction.
Figure 3
Figure 3
Captured segment exchange. (A) Two-step captured segment exchange. In step 1, a BAC containing the region of interest with the desired modifications, an attB site, an FRT site, and white to identify positive transformants, is injected into a stock that has an attP site within targeting distance of the genomic region of interest. In step 2, a FLP/FRT recombination reaction is induced between the newly inserted FRT and an FRT on the homologous chromosome that flanks the opposite site of the region of interest. This results in a new recombinant chromosome containing only the modified region of interest at the endogenous location. (B) One-step captured segment exchange. A BAC containing the modified region of interest and two flanking inverted attB sites is injected into embryos with two attP sites on homologous chromosomes. Upon integration into the genome, the endogenous region of interest is swapped out for the modified region of interest.
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