Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers / 生物工程学报

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

Production of cloned goats by transfer of nuclei from transfected caprine mammary gland epithelial cells / 生物工程学报

In this study, we evaluated the development potential of caprine mammary gland epithelial cells (CMGECs) after transfection and nuclear transfer into enucleated, ovulated oocytes. We first isolated CMGECs from udders of lactating goats which were transfected with expression plasmid for human lacterrin and selected by G418. Then we chose sixteen neomycin resistant lines and induced them with prolactin for the expression of human lactoferrin checked by Western blotting. The donor cells, expressing human lactoferrin of 75 kD, were fused and activated with enucleated ovulated oocytes. Pronuclear-stage reconstructed embryos were transferred into the oviducts of 16 recipient goats. There were fourteen (87.5%), thirteen (81.3%), and ten (62.5%) pregnancies confirmed pregnant by ultrasound on Day 30, 60, and 90, respectively. Three recipients carried the pregnancies to term and delivered one goat each. Nested PCR-RFLP analysis confirmed that all of the kids were clones of the donor cells. These results demonstrated that CMGECs after transfection remain totipotent for nuclear transfer.