Production of Cloned Pigs by Whole-Cell Intracytoplasmic Microinjection1

Abstract

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus)

“…2F) over the 18 number of oocytes used, was significantly improved from 13 to 22% for skin fibroblasts, 19 and from 17 to 34% for cumulus cells when PHA was used. However, the percentage of 20 blastocyst development (29 to 38%) over the number of fused oocytes was not 21 significantly different, regardless of phytohemagglutinin treatments or donor cell type 22 (Table 3).…”

“…2 3 reprogramming of the genome of a differentiated somatic cell nucleus, the donor nucleus 5 must be successfully introduced into the oocyte's cytoplasm, either by direct nuclear 6 injection [6,11], whole cell injection [19], or most commonly, via membrane fusion by 7 electrical stimulation [1,2,10,20,21]. Despite the successes of cloning, however, overall 8 cloning efficiency [3,22,23] has remained low, caused to some extent by the low fusion 9 efficiencies currently achieved between the small somatic donor cell and the recipient 10 oocyte, following somatic NT [1,3,20].…”

“…In addition to the size disparity, the difference in 16 membrane properties among the different somatic cell types considerably affects their 17 fusion efficiency [27]. The development of cloned embryos is largely anomalous, and its 18 inefficiency could be caused by other problems inherent to the oocyte's ability to 19 completely reactivate and/or reprogram an introduced somatic genome during the cloning 20 process [22,[28][29][30]. 21…”

“…In vitro and in vivo development of NT embryos was not harmed by PHA 17 assisted membrane fusion treatment. This efficient cloning technology should be 18 applicable to improve nuclear transfer efficiency in other domestic animals, such as pigs, 19 sheep, rabbits, goats and horses, where electrical fusion is used. The combination of NT 20 and PHA mediated cell fusion could also be applied to the efforts to preserve endangered 21 species.…”

The effect of mitochondrial genome on architectural remodeling and epigenetic reprogramming of donor cell nuclei in mammalian nuclear transfer-derived embryos

“…In this case the only source of exogenous proteins and mRNA transcripts is the nucleoplasm of transplanted karyoplast. Insignificant amounts of perinuclear cytoplasm (perikaryon) remain to interfere with the further embryonic development of mammalian clonal zygotes (Lacham-Kaplan et al, 2000;Prather, 2000;Galli et al, 2002;Roh and Hwang, 2002;Lee et al, 2003c;Samiec, 2004;St. John et al, 2004;Yang et al, 2004).…”