Production of Transgenic-clone Pigs by the Combination of ICSI-mediated Gene Transfer with Somatic Cell Nuclear Transfer

Abstract

The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.

Cloning of Homozygous a1,3-Galactosyltransferase Gene Knock-Out Pigs by Somatic Cell Nuclear Transfer

“…The overall production efficiency of the cloned piglets was 3.1% from the recipients that farrowed or received a Caesarian section (17/547). Cloning efficiency of GalT-KO pigs in these experiments was comparable to that of non-KO pigs that we had reported previously (Kurome et al, 2006;Matsunari et al, 2008b).…”

Effect of Postactivation Treatment with Latrunculin A on <i>In Vitro</i> and <i>In Vivo</i> Development of Cloned Embryos Derived from Kidney Fibroblasts of an Aged Clawn Miniature Boar

“…The use of pig kidney fibroblasts as donor cells for SCNT experiment has been reported by several investigators [8,10,37]. However, to our knowledge, we produced the first cloned miniature piglet derived from kidney cells of an aged adult.…”

“…To date, pigs have been produced by SCNT techniques using donor cells derived from fetuses and young pigs [4][5][6][7][8][9][10][11][12][13]. The use of pig kidney fibroblasts as donor cells for SCNT experiment has been reported by several investigators [8,10,37].…”

“…To date, pigs have been cloned with SCNT using donor cells derived from fetal pigs to pigs several years of age. The efficiency of SCNT is influenced by many factors, including quality of recipient oocytes, type of donor cells, reprogramming of donor nuclei and condition of recipient gilts [4][5][6][7][8][9][10][11][12][13]. In particular, the diploid complement retention is one of the most important factors in improving the developmental ability of SCNT embryos [14,15].…”

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

“…Damage to the sperm plasma membranes causes DNA to associate with important consequences for the transfer of DNA into oocytes (Perry et al 1999). However, sperm pretreatments that markedly disrupt the plasma membrane cause DNA fragmentation (Szczygiel et al 2003), or can damage the sperm nucleus and lead to lower efficiency of embryogenesis following ICSI (Kurome et al 2006, García-Vázquez et al 2009). It is also commonly found that transgenes integrated by passive transgenesis commonly form long concatameric arrays .…”