Figure 1
Figure 1. Comparative mapping and gene targeting of a region of mouse chromosome 5A3 syntenic to a commonly deleted segment of 7q22 identified in human myeloid malignancies. (A) Gene content and order in the human 7q22 CDS compared with that of a region in mouse chromosome 5A3 (based on the Ensembl database and not drawn to scale). (B) Overview of the gene targeting strategy. Targeting at Fbxl13/Lrrc17 resulted in integration of a loxP site (triangle), a neomycin resistance cassette (neo), and the 5′ half of the Hprt minigene (hatched box) at the endogenous (wild-type, wt) Fbxl13 locus, whereas gene targeting at Srpk2 resulted in integration of a loxP site (triangle), a puromycin resistance cassette (puro), and the 3′Hprt-ires-gfp (cross-hatched box) at the endogenous (wt) Srpk2 locus. Expression of Cre recombinase results in cis recombination between the loxP sites contained in the 2 complementary cassettes and reconstitution of a functional Hprt cDNA. The parental cells are resistant to both G418 and puromycin and the recombined cells excise the drug resistance cassettes and are therefore sensitive to both antibiotics. The positions of primers for genotyping analysis (arrowheads) and probes used for Southern blots (solid boxes) are indicated. (C) Confirmation of the deletion of targeted 5A3 region in ES cells. Genomic DNA isolated from doubly targeted ES cells before and after the expression of Cre recombinase were digested with Bsu36I restriction enzyme (Bs), followed by hybridization with probe B (B). Whereas the 15-kb and 9-kb bands diagnostic of the targeted (fl) and wt (+) alleles, respectively, were observed in the doubly targeted ES cells before Cre recombination, a 20-kb band diagnostic of the deleted (del) allele was observed instead of the 15-kb floxed band after Cre expression. (D) Germline transmission of the latent mutant allele was assessed by Southern blotting (top). Tail DNA that is digested with HindIII (H) restriction enzyme and hybridized with probe A (B) shows a 15-kb fragment that corresponds to the wt allele and a 9-kb fragment derived from the fl allele. Confirmation of targeting at 5′ end using PCR genotyping (bottom). Primer fl-reverse (rev) is specific to the targeted allele. The wt allele results in a 376-bp fragment when amplified with wt-forward (for) and wt-rev primers, whereas the targeted allele gives a 245-bp fragment when amplified with wt-for and fl-rev primers. The targeted allele retained the wt amplification product due to duplication of the region of homology contained in the vector when using the insertional targeting strategy.

Comparative mapping and gene targeting of a region of mouse chromosome 5A3 syntenic to a commonly deleted segment of 7q22 identified in human myeloid malignancies. (A) Gene content and order in the human 7q22 CDS compared with that of a region in mouse chromosome 5A3 (based on the Ensembl database and not drawn to scale). (B) Overview of the gene targeting strategy. Targeting at Fbxl13/Lrrc17 resulted in integration of a loxP site (triangle), a neomycin resistance cassette (neo), and the 5′ half of the Hprt minigene (hatched box) at the endogenous (wild-type, wt) Fbxl13 locus, whereas gene targeting at Srpk2 resulted in integration of a loxP site (triangle), a puromycin resistance cassette (puro), and the 3′Hprt-ires-gfp (cross-hatched box) at the endogenous (wt) Srpk2 locus. Expression of Cre recombinase results in cis recombination between the loxP sites contained in the 2 complementary cassettes and reconstitution of a functional Hprt cDNA. The parental cells are resistant to both G418 and puromycin and the recombined cells excise the drug resistance cassettes and are therefore sensitive to both antibiotics. The positions of primers for genotyping analysis (arrowheads) and probes used for Southern blots (solid boxes) are indicated. (C) Confirmation of the deletion of targeted 5A3 region in ES cells. Genomic DNA isolated from doubly targeted ES cells before and after the expression of Cre recombinase were digested with Bsu36I restriction enzyme (Bs), followed by hybridization with probe B (B). Whereas the 15-kb and 9-kb bands diagnostic of the targeted (fl) and wt (+) alleles, respectively, were observed in the doubly targeted ES cells before Cre recombination, a 20-kb band diagnostic of the deleted (del) allele was observed instead of the 15-kb floxed band after Cre expression. (D) Germline transmission of the latent mutant allele was assessed by Southern blotting (top). Tail DNA that is digested with HindIII (H) restriction enzyme and hybridized with probe A (B) shows a 15-kb fragment that corresponds to the wt allele and a 9-kb fragment derived from the fl allele. Confirmation of targeting at 5′ end using PCR genotyping (bottom). Primer fl-reverse (rev) is specific to the targeted allele. The wt allele results in a 376-bp fragment when amplified with wt-forward (for) and wt-rev primers, whereas the targeted allele gives a 245-bp fragment when amplified with wt-for and fl-rev primers. The targeted allele retained the wt amplification product due to duplication of the region of homology contained in the vector when using the insertional targeting strategy.

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