| Summary: | 碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 94 === The aims of this study were to produce porcine embryo in vitro and transfer pCX-EGFP transgene which includes EGFP cDNA under the regulation of a chicken beta-actin promoter and cytomegalovirus enhancer into in vitro and in vivo produced porcine embryos by pronuclear microinjection.
For generation of transgenic pigs, the experiments used the pronuclear embryos derived from in vitro and in vivo production. Ovaries were collected from prepuberty gilts at a local abattoir and cumulus-oocyte complexes (COCs) were isolated from 3-6mm in diameter antral follicles. The COCs were discriminated two groups by oocytes with more (A group) or less than two layers (B group) cumulus cell. COCs were cultured with NCSU-23 medium of in vitro maturation (IVM) for 44-46 hr. After IVM, the oocytes were co-culture with spermatozoa of capacitated treatment for 6-8 hr, then the oocytes were transferred to NCSU-23 medium supplemented with 0.4％ bovine serum albumin for further culture 14 to 20 hr. The results indicated that the percentage of oocytes resume meiosis to metaphase II were 83.28±1.56％ v.s 69.98±6.66％(P<0.05), respectively (P<0.05), average fertilization rate were 68.83±5.5％ v.s 67.18±6.41％, and two-pronucleus formation rate were 19.87±3.44％ v.s 19.68±6.13％.The result of in vitro culture showed that cleavage rate were 57.36±9.76 ％ v.s 47.62±4.02 ％and the percentage of developing to blastocyst stage were 9.04±2.82％v.s 3.95±3.87％. At 16~18hr postinsemination, two-pronucleus embryos provided for pCX-EGFP transgene injection. A total of 66 injected embryos were performed and assessed the EGFP expression by fluorescence microscopy during in vitro culture. Eventually, nineteen out of them express EGFP gene. Furthermore,a total of 47 embryos transferred into oviducts of the four recipient sows which derived from IVP system and injected pCX-EGFP transgen In addition, for the experiment of producing transgenic pigs, totally 265 pronuclear porcine embryos were collected surgically from donor gilts, all of the pCX-EGFP transgene injected embryos were transferred into oviducts of the eight recipient sows. Three out of thirty-six farrowed piglets were confirmed to be as transgenics by PCR and Southern blot analysis. All of them ubiquitously express the EGFP under blue light exposure.
In conclusion, the COCs derived from 3-6 mm in diameter antral follicles have capacity to mature, fertilize and develop to blastocyst in vitro. In the foreseeable future, in vitro production of porcine embryo is possible to provide a large number of porcine embryos for gene transfer. EGFP transgenic pig is useful for establishing pig model of regenerative medicine.
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