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

Abstract

Background: Transgenic cattle carrying multiple genomic modifications have been produced by serial rounds of somatic cell chromatin transfer (cloning) of sequentially genetically targeted somatic cells. However, cloning efficiency tends to decline with the increase of rounds of cloning. It is possible that multiple rounds of cloning compromise the genome integrity or/and introduce epigenetic errors in the resulting cell lines, rendering a decline in cloning. To test these possibilities, we performed 9 high density array Comparative Genomic Hybridization (CGH) experiments to test the genome integrity in 3 independent bovine transgenic cell lineages generated from genetic modification and cloning. Our plan included the control hybridizations (self to self) of the 3 founder cell lines and 6 comparative hybridizations between these founders and their derived cell lines with either high or low cloning efficiencies.

Results: 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).

Conclusions: 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.

Figure 1

Three cell lineages (founders and test cell lines) and their success rates for animal cloning. Live calving rates for the cell lines were calculated by the live calf counts at birth divided by recipient numbers used for embryo transfer as shown in parentheses. Cell lines with 7% or more living rates are indicated as High (H; high calving rate) and those with 0% live calving rate as Low (L; low calving rate). The 3 founder cell lines (F1, F2 and F3) were established from 3 different fetuses (day 40) respectively that were produced by artificial insemination. The 6 test cell lines, except for cell line L3, were derived from 2 rounds genetic modification and somatic cell cloning. L3 line was derived from 3 rounds of genetic modification and somatic cell cloning.

Figure 2

False positive event calls could be due to high GC content. A 5.5 kb variable region (chr25:28829889-28835660) was identified in one control self to self array CGH. GC Percent in 5-Base Windows, Array CGH probe, Gap, RefSeq Gene and Repeat tracks are displayed in Btau_4.0. The GC percent track shows the percentage of G (guanine) and C (cytosine) bases in 5-base windows. The horizontal line at 41.7 in GC percent track represents the genome average of GC%. Probe locations are labeled like CHR25FS027220642 and etc.