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

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

Production of cloned pigs by whole-cell intracytoplasmic microinjection.

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.