Gene targeting and analysis of targeted integrants. (
A) Strategy for replacing the endogenous mouse rhodopsin gene with P23H-hRho-GFP. The targeting vector contains 4.1 kb of upstream and 6.5 kb of downstream sequences from the mouse rhodopsin locus (
dark gray). The P23H-hRho-GFP gene is shown in black, with the exons indicated by
rectangles and introns by
lines and GFP shown in
white. LoxP sites (
inverted triangles) flank the HPRT minigene (
light gray), which was inserted at the boundary between the human and mouse rhodopsin sequences to serve as a positive selectable marker for ES cell integration. The 3′ boundary between human and mouse DNA is marked by a Lox511 site, which is not shown as it played no role in these experiments. We attached the herpes virus TK gene (
white) to the end of the targeting vector to allow selection against random integrants. The portion of the mouse rhodopsin gene in ES cells that was replaced in the targeting is shown in
white. (
B) Southern blot analysis of properly targeted ES cells. To identify ES cells carrying the HPRT-P23H-hRho-GFP gene (HPRT-P23H) in place of the mouse gene, we probed for correctly targeted clones at the 5′ end, by blotting with the 1.1-kb BamHI-
SacI mouse genomic fragment upstream of the 5′-flanking homology used in the targeting vectors. For the 3′ end, we used a 1.0-kb BglII-
EcoRI DNA probe, which is a subfragment of the 6.5-kb 3′-flanking homology used in the targeting vector. Restriction enzymes used to digest genomic DNA are indicated in parentheses. The positions of the relevant BamHI (B) and
HindIII (H) restriction sites are shown in (
A), along with the locations of the hybridization probes (
short lines). The BamHI site on the
left is farther upstream than its position in the diagram would indicate. Correct targeting at the 5′ end is indicated by a 8.9-kb band; correct targeting at the 3′ end is indicated by a 6.7-kb band. Sizes are in kb. (
C) PCR analysis of genomic tail DNA from founder mice carrying the HPRT-P23H allele. We used PCR primers that flanked the GFP gene and amplified both the unmodified mouse allele (+) and the HPRT-P23H allele. Sizes are indicated in kb. (
D) Mating scheme to generate mice that are HPRT-P23H and GDH-9-iCre positive. To eliminate the HPRT minigene, we first bred offspring heterozygous for the HPRT-P23H allele to mice with an iCre transgene driven by the GDF-9 promoter, which is expressed in the female germline,
25 to generate mice that carried both genes (pup 3). Examples of pups with the expected genotypes are shown. (
E) Generation of mice that are P23H-hRho-GFP positive, HPRT negative, and iCre negative. Female HPRT-P23H/GDF-9-iCre mice were crossed to wild type mice and pups were analyzed by PCR to identify those that carried P23H-hRho-GFP (P23H), but lacked HPRT and GDF-9-iCre (pup 1). Examples of mice with the expected genotypes are shown.