Figure 2.

Gene targeting of the human retinoblastoma gene. (A) Overall experimental outline. Steps subsequent to gene targeting that resulted in the selection of clones that bypassed senescence are also indicated. (B) Design of targeting vectors. The genomic loci are shown underneath the vectors and are drawn approximately to scale; black boxes (numbered) represent exons. The grey bars underneath the genomic loci depict the location of Southern hybridization probes. (C) Knockout of one copy of the human RB gene. Southern hybridization was performed with a unique chromosomal probe to the left (5′) of the targeting vector. Further verification of correct chromosomal configuration was performed by PCR of genomic DNA. The summary presents the number of colonies screened and the number of hits recovered. Note that two fibroblast cell strains (SF1 and LF1) were targeted in two independent experiments. SH18 is a derivative of SF1, and LH13 is a derivative of LF1. RB, retinoblastoma.