Production of transgenic blastocyst of sheep by somatic cell cloning |
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Authors: | Xiaorong An Kemian Gou Yongfu Chen |
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Institution: | (1) State Key Laboratory for Agrobiotechnologies, China Agricultural University, 100094 Beijing, China |
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Abstract: | Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP-N1 DNA. Five transgenic
positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total
of 352in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos
were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result
shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage.
Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates
given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly
(P>0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment
indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene
transfection in somatic cells in culture and somatic cell cloning.
These authors contributed equally to this work. |
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Keywords: | sheep granulosa cell nucleus transfer transgenic blastocyst |
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