Phenotypic Effects of Somatic Cell Cloning in the Mouse

Abstract

Although a variety of phenotypes and epigenetic alterations have been reported in animals cloned from somatic cells, the exact nature and consequences of cloning remain unclear. We cloned mice using fresh or short-term cultures of donor cells (cumulus cells, immature Sertoli cells, and fetal or adult fibroblast cells) with defined genetic backgrounds, and then compared the phenotypic and epigenetic characteristics of the cloned mice with those of fertilization-derived control mice. Irrespective of the nucleus-donor cell type, about 50% of the reconstructed embryos developed to the morula/blastocyst stage, but about 90% of these clones showed arrested development between days 5 and 8, shortly after implantation. Most of the clones were alive at term, readily recovered respiration, and did not show any malformations or overgrowths. However, their placentas were two- to threefold larger than those of the controls, due to hyperplasia of the basal (or spongiotrophoblast) layer. Although there was significant suppression of a subset of both imprinted and non-imprinted placental genes, fetal gene suppression was minimal. The seven imprinted genes that we examined were all expressed correctly from the parental alleles. These findings were consistent for every cell type from the midgestation through term stages. Therefore, cloning by nuclear transfer does not perturb the parent-specific imprinting memory that is established during gametogenesis, and the phenotypic and epigenetic effects of cloning are restricted to placental development at the midgestation and term stages. Twelve male mice that were born in a normal manner following nuclear transfer with immature Sertoli cells (B6D2F1 genetic background) were subjected to long-term observation. They died earlier than the genotype-matched controls (50% survival point: 550 days vs. 1028 days, respectively), most probably due to severe pneumonia, which indicates that unexpected phenotypes can appear as a result of the long-term effects of somatic cell cloning.

Review Paper: A Review of the Pathology of Abnormal Placentae of Somatic Cell Nuclear Transfer Clone Pregnancies in Cattle, Sheep, and Mice

“…11,12). 71,76,77,90,91,100 Structural modifications have been reported in mouse placental tissues collected by cesarean section at 18.5 days post coitum (dpc) 100 and after natural delivery at 19.5 dpc 71,76 from SCNT pregnancies with live pups 71,76,100 and, in few cases, with fetuses that died in utero 76 or from respiratory distress after cesarean delivery. 100 Specifically, disorganization of the labyrinthine layer, characterized by irregular branching and dilation of foetal capillaries, and disruption of placental zonation with interdigitations of the boundary between the labyrinthine and basal layers have been described (Figs.…”

“…11,12) 71,76,100 but also are associated with other oocyte micromanipulation techniques, such as microinsemination (sperm or spermatid injection) and pronuclear exchange. 71 Temporal development of SCNT placental lesions is not well characterized. The earliest defective extraembryonic cell lineage detected is the epiblast.…”

“…Genes down-regulated in placentae of SCNT mice include meiosis-expressed gene 1; mesodermspecific transcript; and extraembryonic, spermatogenesis, homebox 1 genes in mid-gestation; and maternally expressed gene 3 and kinase insert domain protein receptor in late gestation. 47,49,71,94,100 Results of gene-targeting studies suggest that the decreased expression of these genes contribute cumulatively to the hyperplastic changes in SCNT placentae. 62,94 Moreover, down-regulation of carboxypeptidase E is associated with placentomegaly in SCNT mice.…”

Placental function in development and disease

“…Because cotyledonary size can undergo a compensatory increase if the number of placentomes is reduced by carunclectomy (Anthony et al 2003), the hypertrophy observed in placentas from clones is likely to be simply a secondary change. In mice, the term placenta in clones is enlarged owing to expansion of the spongiotrophoblast layer, but whether this is a primary or secondary change is unclear (Tanaka et al 2001;Ogura et al 2002;Singh et al 2004).…”

Nuclear reprogramming: what has been done and potential avenues for improvements

“…However, the initial emphasis that has welcomed the production of Dolly the sheep has been progressively quenched in the light of the multitude of developmental defects described in clones [3]. In particular, an emerging insight is that development and function of extra-embryonic membranes is particularly affected by somatic cell nuclear transfer (SCNT) [4,5]. Such placentation dysfunction has dramatic effects on embryonic development and growth, leading to foetal losses in the most severe cases, and also the occurrence of stillbirth and postnatal mortality [6,7], particularly in sheep [8].…”