| The successful production of transgenic animals by somatic cell nuclear transfer (SCNT) has important implications in animal husbandry and biological medicine. However, the efficiency of this technique remains low. In this study, the effects of different breed of recipients, synchronization of estrus time in recipients and activation time of cloned embryo, whether or not ovulation and the number of ovulation on the rate of pregnancy were examined. The genetic relationships of transgenic cloned goats were detected by PCR-SSCP. The integration of the exogenous DNA was detected by PCR, Southern blotting and Tail-PCR. The study established a foundation for further research on transgenic cloned goats.1. The effect of recipient factors on producing hBD3 transgenic cloned goats Total 64 goats were used as the recipients of transgenic cloned embryo transfer, the pregnancy rate was 34.4%(22/64) on the 30 days, which was detected by Brightness mode supersonic diagnostic set. 12 recipients were aborted in the early stage of pregnancy, 10 recipients carried pregnancies to term, the recipient kidding rate was 15.6% (10/64), and finally 14 kids were born. The results showed that: (1) There were no significant differences between the Xinong Shaaneng Dairy Goat and Boer Goat on the pregnancy rates (30.6% vs. 46.7%, P>0.05). (2) The synchronization of estrus time in recipients and the activation time of cloned embryo was termed as 0 day; -1 day (estrus time in recipients was earlier than the activation time of cloned embryo for 1 day); +1 day (estrus time in recipients was later than activation time of the cloned embryo for 1 day). The pregnancy rates had remarkable difference between those groups(53.6% vs. 18.8% or 25%, P<0.05). However there was no significant difference between the -1 day group and +1 day group(18.8% vs. 25%, P>0.05). (3) There was no significant difference between the ovulating group and post-ovulation group (40.9% vs. 44.4%, P>0.05), and significant difference between no ovulation group and the ovulating group or post-ovulation group (6.7% vs. 40.9% or 44.4%, P<0.05). (4) There was no significant difference between the one ovulation site group and two or above two ovulation sites group (33.3% vs. 48.4%, P>0.05). 2. The genetic detection of transgenic cloned goatsThe genetic relationships among transgenic cloned goat, the transgenic donor cell and the recipients were examined by PCR-SSCP. The results showed that the genetic material of the transgenic cloned goat from the transgenic donor cell, and there was no genetic relationship between the transgenic cloned goat and the recipients.3. The Detection of exogenous gene integration in the transgenic cloned goatsThe integration of hBD3 gene in the transgenic cloned goats were examined: (1) The integration of exogenous gene in the transgenic cloned goat was detected by Multiple-PCR, the results showed that hBD3 gene and neo gene had integrated in the genome of the cloned goats. (2) Copy number of the exogenous gene was examinated by Southern blotting, we found that hBD3 gene integrated to the genome of transgene cloned offspring goats with a low copy form. (3) Through the detection of integration sites by Tail-PCR, the blast result showed that the fragment belonged to one part of the sequence in chromosome 4.The results of this study demonstrated that embryo tansfer of transgenic cloned embryo should select the recipients which had the synchronization of recipients estrus time with the cloned embryo activation time; the transgenic cloned goats derived from the transgenic positive cells; all the transgenic cloned goats had integrated with the exogenous hBD3 gene. |
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