攜帶雞β-actin /綠色螢光蛋白轉基因豬之產製與分析

Abstract

本研究旨在利用原核顯微操作技術，將由雞β-肌動蛋白啟動子攜帶綠色螢光蛋白質基因片段與巨細胞病毒促進子之轉基因，注入源自體內與體外受精所獲得之原核時期豬胚並經胚移置於受胚母豬。以期產製全身表現綠色螢光蛋白質之基因轉殖豬，做為生物醫學相關研究之動物平台。 為達此目的，本研究利用體外生產系統與外科手術取得原核時期的受精卵，供作基因顯微注射用。 源自屠宰場獲得發身前之母豬卵巢，選擇大小為3-6mm之濾泡，分離卵丘卵母複合體(cumulus-oocyte complexes, COCs)。並將取得卵丘卵母複合體依據包覆卵母細胞外圍之卵丘細胞層數分為具有兩層以上卵丘細胞(A組)與少於兩層以上卵丘細胞(B組)之兩組試驗組。經應用NCSU-23體外培養液，於 39℃、5％CO2及100％空氣與100％相對濕度等條件下，進行體外成熟作用44-46小時。培養終了平均分別可獲得83.28±1.56％ 與69.98±6.66％之成熟卵母細胞。進ㄧ歩將成熟卵母細胞與業經體外獲能處理4小時之精子共培養6至 8小時後，移至體外培養液中培養14~16小時，經位向差顯微鏡觀察受精率與原核形成比例，結果兩組平均受精率分別為68.83±5.5％ 與67.18±6.41％；可發育形成雌雄原核之百分率分別為19.87±3.44％ 與19.68±6.13％。由體外成熟、受精及受精卵於體外培養7日後，其卵裂率分別為57.36±9.76 ％與47.62±4.02 ％，而其可發育至囊胚期豬胚之百分率則為9.04±2.82％與3.95±3.87％。至於豬胚外源性基因之顯微注射係受精後16至18小時予以進行，並經體外培養７日後，以螢光顯微鏡觀察豬胚表現綠色螢光情形。總計完成66個豬胚顯微操作，培養終了計有19顆豬胚表現綠色螢光。透過實驗證實，源自體外成熟、受精、培養及顯微注射外源基因之豬胚，能在體外順利發育並表現綠色螢光。因此進一步試驗，將源自體外成熟、受精、培養及顯微注射外源基因之豬胚，經胚移置於受胚豬兩側輸卵管中，觀其在體內發育的潛能，總計47 顆豬胚經胚移置於四頭受胚母豬。 利用源自供胚母豬經外科手術收集之豬胚所進行之產製螢光豬試驗，總計收集265 顆處於原核時期之豬胚，業經基因顯微注射後，移置八頭受胚母豬之兩側輸卵管中。經懷孕期滿，共36頭仔豬順利分娩，而其中有三頭仔豬其基因組DNA經聚合酶鏈鎖反應(PCR)與南方吸漬法(Southern blot)分析後，證實三隻均帶有外源性轉殖基因。並於藍光激發下，其全身性發射出綠色螢光。 綜合上述，可知源自半徑大小為3-6mm濾泡之卵丘卵母複合體經體外成熟、受精作用後所獲得之豬胚，可順利發育至囊胚時期。經此過程所獲得之原核期豬胚經顯微操作後，可順利表現綠色螢光蛋白質。可以預見，未來透過體外培養技術，將可獲得大量可供作基因轉殖之豬胚，降低產製基因轉殖豬之成本。而透過上述試驗所獲得之綠色螢光基因轉殖豬將有助於協助建立再生醫學之大型動物模式之研究。

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.