Figure 1.

LoxP targeting of M6P/IGF2R exon 10. A: Generation of the M6P/IGF2R LoxP targeting construct involved the cloning of 7-kb of the mouse genomic DNA encompassing exons 8 through 11 and flanking intronic sequence into an appropriate LoxP vector. The M6P/IGF2R LoxP targeting construct was then transfected into Sv129 ES cells. Homologous recombinants were selected for using G418 (neomycin) and confirmed by PCR. B: Cre-recombinase plasmid (1 μg) controlled from the constitutively active CMV promoter was transfected into the ES cells which were then grown in the presence of gancyclovir to select for recombinants that had lost the TK/Neo cassette; PCR was used to confirm loss of the TK/Neo cassette. Recombinant ES cells were injected into the blastocoele cavity of the 3.5-day-old C57Bl/6J embryos before implantation into pseudo-pregnant mice. C: Expression of Cre-recombinase in mice with a floxed exon 10 results in its excision generating a M6P/IGF2R protein that encodes for a truncated receptor lacking the M6P and IGF2 extracellular binding domains. DT, TK, and Neo enable both positive and negative selection.