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1、慢病毒介導HLA-G轉(zhuǎn)基因小鼠的建立王露露，劉勤，周曉楊，劉昌峨，王勇，魏泓（400038重慶，第三軍醫(yī)大學基礎(chǔ)醫(yī)學部實驗動物學教研室）[摘要]目的以慢病毒作為載體建立HLA-G轉(zhuǎn)基因小鼠，為研究HLA-G與移植免疫提供模型動物。方法利用FUW載體，構(gòu)建含有HLA-G編碼序列的慢病毒表達載體FUW-HLA-G。用磷酸鈣沉淀法將包裝質(zhì)粒psPAX2、PMD2.G與重組慢病毒載體質(zhì)粒FUW-HLA-G共轉(zhuǎn)染293FT細胞，包裝成慢病毒。通過同步轉(zhuǎn)染含有綠色熒光標記基因的慢病毒質(zhì)粒FUGW為參照，測定FUW-HLA-G病毒液的滴度。用顯微注射法將濃縮后的病毒液注射至FVB/N小鼠1-細胞期胚胎
2、透明帶下，建立HLA-G轉(zhuǎn)基因小鼠。PCR鑒定轉(zhuǎn)基因小鼠的基因整合，用RT-PCR檢測轉(zhuǎn)基因在轉(zhuǎn)錄水平的表達，用Westernblot檢測HLA-G蛋白的表達。結(jié)果載體BamHI、EcoRI雙酶切結(jié)果和測序結(jié)果顯示重組的慢病毒質(zhì)粒與所設(shè)計的序列一致。濃縮和純化后的病毒液滴度達到109TU/ml以上，符合用慢病毒制備轉(zhuǎn)基因小鼠的要求。PCR篩選出陽性鼠6只，RT-PCR與Westernblot檢測結(jié)果表明HLA-G基因在F0和F1代轉(zhuǎn)基因小鼠中均有表達。結(jié)論本實驗通過慢病毒介導的基因轉(zhuǎn)移，成功建立了表達人HLA-G蛋白的轉(zhuǎn)基因小鼠，為研究HLA-G的功能及其在器官或組織移植中的效應(yīng)提供了動
3、物模型。[關(guān)鍵詞]HLA-G；慢病毒；轉(zhuǎn)基因小鼠[中圖法分類號]R-332；R373；R392.4[文獻標志碼]AGenerationofHLA-GtransgeniemicebylentiviraltransgenesisWangLu1u,LiuQin,ZhouXiaoyang,LiuChang’e,WangYong,WeiHong(DepartmentofLaboratoryAnimalScience,CollegeofMedicine,ThirdMilitaryMedicalUniversity,Chongqing,400038,China)[Abstract]0bjectiveT
4、oestablishHLA-Gtransgeniemicebylentiviraltransgenesis,andtherebytoprovideananimalmodeltoinvestigatetheHLA-G’simpactsontissueororgantransplantation.MethodsThetransgenevectorwasconstructedbyinsertingtheHLA-GencodingfragmentintoalentiviralvectorFUW.Theresultedrecombinantlentiviralvector,FUW-HLA-G,wa
5、spackagedintolentiviralparticlesbyco-transfectingtherecombinantlentiviralvectorinto293FTcellsalongwithtwopackagingvectors,psPAX2andpMD2.G.Theviralsuspensionwasconcentratedby“two-step”high-speedcentrifugation.ForaccuratetitrationofFUW-HLA-Glentiviralparticles,anotherlentiviralvectorcontainingamark
6、ergeneeGFPwaspackagedandconcentratedinparallel.Transgenicmiceweregeneratedbyinjectingtheconcentratedviralsuspensionintoperivitellinespaceofmicemurine1-celleggs.ThetransgenicfoundermicewerescreenedbyPCRandtransgeneexpressionwasdetectedbyRT-PCRandWesternblot.ResultsDoublerestrictivedigestionandsequ
7、encingconfirmedthattherecombinantlentiviralvectorFUW-HLA-Gwascorrectlyconstructed.ThebiologicaltitresoftheFUW-HLA-Gvirusconcentratesweredetectedtobenolessthan1×109TU/ml,whichwassufficientforlentiviraltransgenesisinmamm
