| The successful production of transgenic animals by somatic cell nuclear transfer (SCNT) has important implications in agriculture and medicine. However, the efficiency of this technique remains low and cloned animals had been found to have high rates of abnormalities. Moreover, the procedure of transgenic nuclear transfer is complicated. Few scientific research groups can produce a mass of transgenic animals by this technique. In order to producing transgenic dairy goats resistant to mastitis, the present study optimized the procedure of goat SCNT and the production of transgenic donor cells.1. We evaluated effects of the interval between fusion and activation (1-5 h), cytochalasin B treatment after electrofusion, embryo culture conditions, stages of embryo transfer and number of transferred embryos on the in vivo and in vitro development of cloned goat embryos. The majority of the reconstructed embryos exhibited condensed chromosomes and metaphase-like chromosomes at 2 and 3 h after fusion, and cleavage and blastocyst rates from those two groups were higher than those of embryos activated at 1, 4 and 5 h after fusion (P < 0.05). CB treatment between fusion and activation improved in vitro and in vivo development of nuclear transfer (NT) goat embryos by reducing the fragmentation rate(P < 0.05). Although there were no significant differences in NT efficiency, the pregnancy rate and kids born per recipient were increased by transfer of 20 or 30 embryos per recipient compared to 10 embryos group. These results suggest that CB treatment for 2 to 3 h between fusion and activation is an efficient method for generating cloned goats by somatic cell nuclear transfer. In addition, increasing the number of embryos transferred to each recipient resulted in more live offspring from fewer recipients.2. We investigated the proliferative capacity, chromosome stability, cell cycle and apoptosis, transfection efficiency and efficiency of producing HLY transgenic goats. The results showed that the proliferative capacity and chromosome stability: GFF1, GFF2>GFF4, GFF3; percentage of G1/G0 phase cells: GFF4>GFF3, GFF5>GFF2>GFF1; percentage of apoptosis cells: GFF4<GFF1, GFF5<GFF3<GFF2; transfection efficiency: GFF2, GFF1>GFF5, GFF4, GFF3; in vitro development of cloned embryos from different transgenic cell lines: F2C11≥F3C1, F1C3, F1C7, F3C2, F2C2>F5C3, F5C3; in vivo development of transgenic cloned embryos: F1C3, F1C7 > F3C1, F3C2. These results indicated that transfection efficiency are higher in the GFF cell lines with high proliferative capacity and chromosome stability, and that not only different cell lines, but also different clones derived from one primary cell line, dramatically affect the developmental potential of NT embryos. So in order to produce viable transgenic cloned animals, the donor cells should be prudently selected.3. This study was conducted to producing HBD3 transgenic goats by SCNT. Seven colony cell lines were established, two of them were used as donor cells. The pregnancy rate on 30 d was 40.5% (32/79), 21.5% (17/79) pregnant goats carried the conceptus to term. Seventeen pregnant goats give birth to 26 kids, the kidding rate was 32.9%(26/79), multiple pregnancy rate was 35.3%(6/17), NT efficiency was 1.4 %. Nine alive kids were transgenic goats screened by PCR and southern blotting.4. The expression of HBD3 was detected by Tricine SDS-PAGE and Western blotting, and the development of recloned embryos from transgenic goat mammary gland epithelial cells (TGMEC) were examined. The results showed that TGMEC can express HBD3 protein, and that the development rates between TGMEC and control group were not significant different. However, the fusion rate of embryos from both TGMEC and control group were lower than that of GFF. The pregnancy rate was 20%. This results indicant that the development capacity of transgenic embryos could not be improved by recloned. |
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