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Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development

Wang, S., Sun, X., Ding, F., Zhang, K., Zhao, R., Li, S., Li, R., Tang, B., Zhang, L., Liu, Y., Li, J., Gao, F., Wang, H., Wang, L., Dai, Y., Li, N.
Theriogenology 2009 v.72 no.4 pp. 535-541
genetic transformation, pregnancy rate, green fluorescent protein, cattle, polymerase chain reaction, calving, transgenic animals, gene expression, blastocyst, reporter genes, fibroblasts, genetic markers, cell cleavage
Introduction of selectable marker genes to transgenic animals could create an inconvenience to further research and may exaggerate public concerns regarding biological safety. The objective of the current study was to excise loxP flanked neoR in transgenic cloned cattle by transient expression of Cre recombinase. Green fluorescent protein gene (GFP) was incorporated to monitor Cre expression; therefore, Cre-expressed cells could be selected indirectly by fluorescence-activated cell sorting (FACS). The neoR was removed and Cre expressed transiently in GFP-positive colonies; excision of neoR was confirmed by single-blastocyst PCR in recloned blastocysts, with neoR-free fibroblast cells as donors. There was no difference (P>0.05) in rates of cleavage (76.0% vs. 68.8%) or blastocyst formation (56.6% vs. 52.9%) between recloned embryos with neoR-free or neoR-included donors. The differential staining of recloned blastocysts were similar (P >0.05) in terms of total cell number (124 vs. 122) and the ratio of ICM (Inner Cell Mass) to the total cell number (38.1% vs. 38.2%). Furthermore, pregnancy and calving rates were not different (P>0.05) from those of the control. In conclusion, we successfully excised neoR from transgenic cloned cattle; the manipulation did not affect the developmental competence of recloned preimplantation embryos. This approach should benefit bioreactor and transgenic research in livestock.