Assessment of genome integrity with array CGH in cattle transgenic cell lines produced by homologous recombination and somatic cell cloning

We detected similar amounts of differences between the control hybridizations (8, 13 and 39 differences) and the comparative analyses of both "high" and "low" cell lines (ranging from 7 to 57 with a mean of ~20). Almost 75% of the large differences (>10 kb) and about 45% of all differences shared the same type (loss or gain) and were located in nearby genomic regions across hybridizations. Therefore, it is likely that they were not true differences but caused by systematic factors associated with local genomic features (e.g. GC contents).Our findings reveal that large copy number variations are less likely to arise during genetic targeting and serial rounds of cloning, fortifying the notion that epigenetic errors introduced from serial cloning may be responsible for the cloning efficiency decline.As embryonic stem cells are not available in the bovine species, somatic cells have been used for genetic modifications, and transgenic cattle have been produced from such genetically modified somatic cells by animal cloning. However, because primary somatic cells have limited life span and inevitably become senescent following DNA transfection and selection in cell culture, it is impossible to perform any further genetic modifications in these cells. Because of such, transgenic cattle with a desired genotype that requires more than one genetic targeting event, such as homozygous deletion of the two alleles of a gene, cannot be produced. To overcome such limitations, a novel sequential genetic modification strategy in bovine somatic cells, for producing extensively genetically modified cattle, has been developed [1]. This process involves a serial round of genetic targeting events, each followed by cloning to rejuvenate the genetically modified somatic cells (to rescue them from senescence) for the next round of genetic targeting. Such genetically modified somatic cells are then subjected to a final round of cloning for producing transgenic animals with the desired genotype