Recloning Of Transgenic Cloned Goats

Transgenic animal research is one of the most active areas in biology. The technique of somatic cell nuclear transfer (SCNT) has many advantages when used in transgenic animal production, such as increasing the number of transgenic animal rapidly without losing foreign gene. Serial animal cloning would provide an excellent model to study the effects of progressively accumulated somatic mutations on development, health and reproductive performance.Cumulus cells and ear-tip fibroblasts from transgenic cloned goats were used as donor cells in this study. Metaphase II (MII) oocytes were collected from oviduct of superovulated donor goats and enucleated as recipient cytoplasts. A single donor cell was injected into the perivitelline space of enucleated oocyte and fusion was induced with electric pulses. Reconstructed oocytes were activated using ionomycin (5 M,5min) and 6-dimethylaminopurine (6-DMAP,2mM,5h). Results,(l)The fusion rate of cumulus cells was 49.3%, fibroblasts was 56.2%. (2) The proportion of morula and blastocysts of cumulus cells was 25%, fibroblasts was 32.7%. (3) The success rate (offspring VS. reconstructed oocytes) of cumulus cells was 1.1%, fibroblasts was 1.3%. (4)Five recloned goats were born and two were survived. (5) PCR-RFLP analysis confirmed that all cloned offspring were derived from the donor goats.Polymerase chain reaction (PCR) analysis showed that all of recloned goats inherited donor goats' foreign gene~neor gene. In order to analyse the expression of neor gene, ear-tip fibroblasts were separated from donor goats and recloned goats and suffered selection of 800 g/ml G418. The results showed that the expression patterns of neorgene were different in different goats' cells cultured in vitro.It is concluded ,(1) both kinds of somatic cells from transgenic cloned goats can be defifferentiated and reprogrammed in MII oocytes cytoplasts and have totipotent potentialof development, the success rate is comparable with normal goats' somatic cell nuclear transfer. (2)Foreign gene can be passed down during nuclear transfer but the expression patterns are different in different goats' cells cultured in vitro.