Production of cloned mice from embryonic stem cells arrested at metaphase

Abstract

In mammals, cloned individuals can be produced from somatic cells. The combined use of gene targeting in embryonic stem cells and cloning contributes to the investigation of gene function in mammals. However, one of the major limitations to cloning is the low viability of cloned embryos, leading typically to high rates of pre- and postnatal death. The present study investigated whether cloning efficiency is influenced by the procedural differences involved in using transfected embryonic stem cells arrested at M phase for cloning by both single and serial transfer. In contrast to a previous study, in which fibroblasts were used, in the present study using embryonic stem cells there was no difference in the rate of production of cloned pups after the use of a single or serial nuclear transfer, although the proportion of blastocysts (70% versus 51%) was significantly higher (P < 0.001) after serial nuclear transfer. After embryo transfer of 445 blastocysts, 218 (49%) implanted and 27 (6% of blastocysts transferred) live pups were born. Of these 27 pups, 23 developed to adults of apparently normal fertility. Of these adults, 39% (n = 9) were derived from targeted embryonic stem cells, which is similar to the proportion of targeted embryonic stem cells in the population used for cloning. This study showed that cloning with embryonic stem cells is a viable procedure resulting in the production of transgenic cloned adults.

Differential Developmental Ability of Embryos Cloned from Tissue-Specific Stem Cells

“…This donor cell-dependent difference may arise because of the undifferentiated status of the donor genome; neonatal Sertoli cells are small, round, immature cells, unlike the large cells in the mature testis. This assumption is consistent with evidence showing that undifferentiated embryonic stem (ES) cells are the best donor cells for mouse cloning, leading to approximately 20% birth rates per embryo transfer in optimal conditions [5][6][7]. However, we found previously that hematopoietic stem cells (HSCs), the most undifferentiated cells of the hematopoietic lineage, are very inefficient donor cells compared with other differentiated cells of the same lineage [8,9].…”

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.…”

Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring

“…These include identifying a better donor cell type or cell cycle stage , treating donor cells with pharmacological agents to alter their epigenetic marks (Enright et al 2003, Kishigami et al 2006, fusing transiently permeabilized cells containing artificially condensed chromatin (Sullivan et al 2004), using serial NT (Ono et al 2001a(Ono et al , 2001b, or aggregating SCNT embryos (Boiani et al 2003). Here, we have focused on improving another step of the cloning procedure, namely, the method of SCNT reconstruct activation.…”