Chimeric Animals and GermlineTransmission

Gene targeting by homologous recombination in pleuripotent embryonic stem (ES) cells provides a powerful tool to introduce specific mutations in the genome of intact animal (1 -5 ). It, therefore, allows to unravel the function of genes that control development and differentiation in the organism. Generally, a gene targeting cassette that contains positive-negative selection markers is prepared in which an exon of the target gene is interrupted by the gene for neomycin resistance, which serves as a positive selection marker (6 -8 ). A herpes simplex virus thymidine kinase (HSV TK) gene is fused at either one or both ends of the genomic sequences for negative selection. The targeting cassette is introduced into ES cells, and the cells in which the endogenous gene is disrupted are selected with G418 and gancyclovir or FIAU (6 ,8 ). The cells in which the DNA is inserted randomly will die as a result of the incorporation of gancyclovir or FIAU, the thymidine analogs, by the thymidine kinase gene, which blocks the DNA synthesis. The selected ES cell clones are then used to prepare chimeric animals for germline transmission.