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.

Fig. 1.

Graphic representation of mutation frequencies in donor cells, male gametes, cloned fetuses, and natural fetuses. Frequencies of mutations detected in the lacI transgene in each donor cell type used for cloning were 1.01 +/- 0.20 × 10⁻⁵ in fetal brain (FB) cells, 2.54 +/-0.57 × 10⁻⁵ in adult cumulus (AC) cells, and 4.04 +/- 0.59 × 10⁻⁵ in adult skin (AS) cells, as compared to 0.78 ± 0.35 × 10⁻⁵ in adult spermatozoa. Mutation frequencies in cloned fetuses were 0.99 ± 0.23 × 10⁻⁵ in fetuses cloned from fetal brain cells, 0.99 +/- 0.24 × 10⁻⁵ in fetuses cloned from adult cumulus cells, and 1.03 +/- 0.25 × 10⁻⁵ in fetuses cloned from adult skin cells, as compared to 0.87 +/- 0.19 × 10⁻⁵ in naturally conceived fetuses. These results are shown in tabular form in Table S1. Error bars represent standard errors.

Fig. 2.

Graphic representation of mutation types in donor cells, cloned fetuses, and natural fetuses. Percentages of types of mutations detected in the lacI transgene in each donor cell type and in fetuses cloned from each donor cell type are represented, as are those for natural fetuses. Transitions = CG <-> TA; Transversions = CG <-> GC or TA <-> AT; Ins/Del = small (≤ 2–3 bp) insertions or deletions; DBS = double base substitutions. FB = fetal brain cells; AC = adult cumulus cells; AS = adult skin cells. Unprocessed numbers, specific percentages, and the specific location and sequence change of each mutation or mutation type in each cell type are shown in tabular form in Table S2 and Table S3. Error bars represent 95% confidence intervals.

Fig. 3.

A bottleneck effect limits transmission of acquired mutations to cloned offspring. A population of donor somatic cells is shown, with a small proportion carrying acquired mutations at a single assayable locus (filled black circles). The potential for transmission of such mutations to cloned offspring is severely limited by a bottleneck effect imposed by the fact that only a single donor cell is used to generate each cloned fetus. However, new mutations can then accrue spontaneously during development of the cloned fetus as indicated by the white circle.