Supplementary Figure Legend.
Schematic of Homologous Recombination at the endogenous DmWWOX gene.
We performed “ends-in” targeting essentially as described in Rong et al. 2002.
(a) 5kb genomic region of DmWWOX was amplified from wild-type genomic DNA and site directed mutagenesis was used to introduce mutations 24 bp downstream of the start codon. These mutations encode two premature stop codons, a +1 frame shift and a diagnostic SalI site. In addition, an I-SceIsite necessary for the targeting process was introduced into the third intron. This mutated fragment was cloned into the pTV2 vector, transformed into wild-type(w1118) flies and used to generate donor lines for gene targeting. (b) FLP-induced recombination at FRT sites in donor flies promotes excision and circularisation resulting in extrachromosomal DNA. This is linearised by digestion with I-SceIand the resultant double strand break facilitates strand invasion at the homologous DmWWOX gene, and results in the generation of insertion lines. (c) A “Type II” insertion event is shown with two copies of the DmWWOX gene separated by the white eye colour marker gene. The engineered mutations are carried in the 3’ copy of the DmWWOX gene. (d) The presence of an I-CreIsite provides a site for generation of a double strand break, such that the two copies of DmWWOX can become aligned and by recombination, reduce the insertion event to a single copy of DmWWOX. Depending on where recombination has occured these reduced flies may have regenerated the wild-type DmWWOX gene or alternatively may have incorporated the engineered mutations into the endogenous gene, thus generating DmWWOX1.
Methods
Two donor lines of the mutated DmWWOX genomic region in the pTV2 targeting vector were established by standard transformation procedures into a wild-type (w1118 ) background and mapped to the third chromosome. Virgins of these red eyed (due to the presence of the whitemarker gene) stocks were crossed to hsFLP,hs-SceI/TM3Ser males and the resulting 48-72hr larvae were heat-shocked for 2hr at 37oC. Virgin females with mosaic eyes were collected and crossed to ey-FLP males. Progeny from this cross were screened for any flies with a solid red eye indicating they now carry the marker gene, indicative of an insertion event. This differentiates them from the original donor lines which would continue to produce mosaic eyes in the presence of ey-FLP. The number of flies screened was estimated by weight (1 fly is approx. 1 mg) and 5 insertion events were detected out of approximately 193,000 flies scored. Four of these insertion lines mapped to the second chromosome (the location of the endogenous DmWWOX gene) whilst the fifth mapped to the X chromosome and was discarded. To perform the reduction, each of the four insertion lines was crossed to hs-CreI/TM6B and 48-72hr larvae were heat-shocked for 1hr at 36oC. Males with mosaic eyes were obtained with two of the insertion lines, indicating that the marker gene had been lost in some cells. White eyed progeny were obtained from multiple mosaic males in one of these lines, suggesting that the reduction had occurred in the germline. These white eyed males were crossed to balancers to make stocks and homozygous flies were then tested by PCR and sequencing for the presence of the engineered mutations.
The pTV2 targeting vector and fly lines for the targeting were a kind gift from K. Golic. Full genotypes of these stocks are w1118; [70FLP][70I-SceI]/TM3Ser (hsFLP,hs-SceI/TM3Ser) and w1118; v,{v+,70I-CreI]1A/TM6B (hsI-CreI/TM6B).
yw, FRT19A; ey-FLP (ey-FLP) and w1118 (wild-type) was obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN).