Production of dairy goat embryos, by nuclear transfer, transgenic for human acid o-glucosidase

摘要

Expression of recombinant human lysosomal acid o-glucosidase (hGCase) by a transgenic animal bioreactor, using somatic cell nuclear transfer (SCNT), would decrease the cost of producing this product. The objective was to establish an effective procedure to prepare hGCase transgenic donor cells and nuclear transfer (NT) embryos to produce hGCase protein in the Saanen dairy goat mammary gland. A mammary-specific expression vector for hGCase was constructed and transfected into HC-11 mammary epithelial cells for bioactivity analysis in vitro; mRNA transcripts and hGCase protein were correctly expressed in transfected HC-11 cells. The hGCase gene was then introduced into fetal fibroblasts (from dairy goats) to prepare competent transgenic donor cells. Transgenic fibroblast clones from a single round of transfection were reliably isolated by 96-well cell culture plates and screened with PCR amplification and chromosomal counting (66.8%). Dairy goat cloned embryos were produced from these hGCase fetal cells by SCNT, the hGCase transgene was successfully detected in these embryos, and there were similar rates (P>0.05) of fusion (83.3% vs. 77.8%), cleavage (89.1% vs. 90.9%), and development to the morula/blastocyst stages (36.4% vs. 38.9%) between NT embryos using transgenic fetal fibroblasts and non-transfected control cells. Moreover, 98 well-developed reconstructed embryos derived from transgenic cells were transferred to 16 recipients; pregnancy was confirmed at 40 d in two goats. Therefore, we achieved functional expression of hGCase in mammary gland cells and normal development to Day 40 of cloned embryos carrying the hGCase gene.