Epigenetic regulation of genetic integrity is reprogrammed during cloning

Abstract

Cloning by somatic cell nuclear transfer (SCNT) circumvents processes that normally function during gametogenesis to prepare the gamete genomes to support development of new progeny following fertilization. One such process is enhanced maintenance of genetic integrity in germ cells, such that germ cells typically carry fewer spontaneously acquired mutations than somatic cells in the same individual. Thus, embryos produced from somatic cells by SCNT could directly inherit more mutations than naturally conceived embryos. Alternatively, they could inherit epigenetic programming that predisposes more rapid accumulation of de novo mutations during development. We used a transgenic mouse system to test these possibilities by producing cloned midgestation mouse fetuses from three different donor somatic cell types carrying significantly different initial frequencies of spontaneous mutations. We found that on an individual locus basis, mutations acquired spontaneously in a population of donor somatic cells are not likely to be propagated to cloned embryos by SCNT. In addition, we found that the rate of accumulation of spontaneous mutations was similar in fetuses produced by either natural conception or cloning, indicating that cloned fetuses do not acquire mutations more rapidly than naturally conceived fetuses. These results represent the first direct demonstration that the process of cloning by SCNT does not lead to an increase in the frequency of point mutations. These results also demonstrate that epigenetic mechanisms normally contribute to the regulation of genetic integrity in a tissue-specific manner, and that these mechanisms are subject to reprogramming during cloning. epigenetic reprogramming ͉ mutagenesis

Enhanced Genetic Integrity in Mouse Germ Cells1

“…Male and female somatic tissues were collected from both sexes at 15.5 dpc and from female pups at 2-6 dpp, frozen in liquid nitrogen, and stored at À808C. Genomic DNA was isolated from each germ cell population and from somatic tissues as previously described [35,36]. Frozen germ cells were resuspended in 50 ll of ice-cold lysis buffer and pipetted up and down several times to lyse the cells and liberate genomic DNA.…”

“…The lambda shuttle vector containing the lacI mutation reporter was selectively recovered from genomic DNA using Transpack packaging extract (Stratagene/Agilent) according to the manufacturer's instructions as previously described [35,36]. Packaged phage were mixed with SCS-8 E coli strain cells (Stratagene/Agilent Technologies) and plated at 17 500 plaque-forming units per 25 3 25 cm NZY agar (Stratagene/Agilent Technologies) assay tray.…”

“…These primers, which would normally be expected to amplify a 3949-bp product from the unmutated lambda shuttle vector transgene were able to amplify a 2011-bp PCR product from mutants that carried a 1938-bp deletion that ablated the binding site for the lacI AF primer, thereby explaining the inability to amplify a product from these mutants by using the lacI AF and lacI AR primers. As with other incidences of recovery of more than one mutant plaque from the same original cell sample carrying the same mutation at the same position in the transgene, this multiplicity was assumed to be caused by clonal expansion of a single original mutation and so was scored as a single mutation when we determined the mutation frequency in that cell sample regardless of how many copies of the same mutant plaque were recovered, as previously described [35,36,[38][39][40][41][42].…”

“…We also purified type T1 prospermatogonia from male fetuses at 15.5 dpc. For each sample, we determined the frequency of mutations in the lacI reporter gene as described previously [15,35,36]. The spontaneous mutation frequencies determined for male and female germ cells at 15.5 dpc (0.99 3 10 À5 and 0.82 3 10 À5 , respectively) were not significantly different from one another (P ¼ 0.75) but were significantly lower than the frequencies found in male and female somatic cells at this same stage (2.82 3 10 À5 and 2.91 3 10 À5 , respectively; P ¼ 0.05) ( Table 2).…”

Further Advice on the Implications of Animal Cloning (SCNT)

“…It has been shown in mouse foetuses that the process of SCNT does not lead to an increase in the frequency of point mutations, and that the clone foetuses accumulated spontaneous mutations at a rate similar to that in foetuses produced by natural conception (Murphey et al, 2009). Moreover, mutations in a marker gene were corrected similarly in early embryos generated by cloning or natural fertilization.…”