Deletion Of The Selectable Drug-resistant Gene In Transgenic Goats Expressing Human Lysozyme

Transgenic animals are those that have been genetically modified and can transfer the exogenous genes to their offspring. Selectable drug-resistant genes are often used during the production of these animals. These sequences usually derive from the virus or bacteria. They could be harmful for the animal health and reduce the expression level of the transgenes. In this paper, we explore the feasibility of deleting selectable genes by Cre/loxP system in goat ear fibroblasts (GEF) integrated with loxP-neo-tk-loxP(LNKL) cassette.First, we culture GEF-BLG—1 and GEF-BLG—2 in vitro. These cells are isolated from the transgenic goats expressing recombinant human lysozyme (rhLYZ). The selectable genes could be deleted by transient expression of Cre recombinase in these cells, but the efficiency is relatively low. Only 4 cell clones were found to contain no LNKL cassette in 515 cell clones analyzed by PCR. In addition, all the four cell clones were senesced and could not be used for animal cloning.Then, we explore the possibility of deleting selectable genes by protein transduction. BL21(DE3) transformed with the pET-cre vector is cultured in 37℃for 3h and induced by adding 1mM IPTG. CRETAT protein was highly expressed and could contain 48.6% of the total proteins. CRETAT proteins were purified by chromatography using XK-16-SP sepharoseTM followed by XK-26 source 15s. The purified CRETAT fusion proteins have biological activity by in vitro test assay.CRETAT fusion proteins were introduced into GEF-BLG—1 or GEF-BLG—2 cells by adding them directly into the culture medium. After several days, the cell colonies were isolated and analyzed G418 sensitive experiment, PCR amplification and Southern-blotting. We successfully obtain several cell clones that contained lysozyme gene but no selectable genes. The rate of the deletion is 42%. In addition, the cells did not show any signs of senescence and their shape is normal.These cell clones were used in nuclear transfer and the blastula development rate of these cells is 0.30,which is similar to the rate of GEF-BLG—2 cells (0.09).In conclusion, we establish a safe and effective system which can delete the selectable drug-resistant gene in transgenic goats. Cre/loxP system by protein transduction can be used to effectively delete the DNA sequence flanked by loxP in transgenic animal. The characteristic of the method is efficient, easy to handle, and can be used in many different cells. Up to date, there is no similar report.