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制备人 β2m转基因小鼠 ,研究HLA B2 70 4基因的表达 .应用显微注射将人 β2m基因注入C5 7BL 6×昆明鼠和昆明鼠×昆明鼠F1代受精卵 .出生动物及其后代经PCR筛选 ,采用斑点杂交和Southern杂交对阳性鼠基因组DNA标本进行进一步鉴定和测定整合拷贝数 ,利用RT PCR检测阳性鼠中人 β2m转基因的表达 .6只原代仔鼠及 7只它们的下一代鼠 (F1)带有人 β2m基因 .由微注射基因后移卵出生的 86只小鼠中 ,C5 7BL 6×昆明鼠杂交仔鼠 35只 ,其中 4只阳性 (11 4 % ) ,昆明鼠×昆明鼠杂交仔鼠 5 1只 ,其中 2只阳性 (3 9% ) ,含有人 β2m基因的原代鼠×昆明鼠杂交仔鼠 2 0只 ,其中 7只阳性 .整合的转基因均为单拷贝 .Southern杂交证实上述阳性鼠确有转基因整合 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有人β2m转基因mRNA的表达 .在转基因动物制备中 ,C5 7BL 6×昆明鼠F1代明显优于昆明鼠×昆明鼠F1代 .与人HLA B2 70 4基因相比 ,人 β2m基因不易整合 ,其整合率与整合拷贝数均较低 .得到的人 β2m转基因小鼠能够将人 β2m基困传给下一代 ,并可与人HLA B2 70 4转基因鼠交配 ,研究它的致病性
Human β2m transgenic mice were prepared and the expression of HLA B2 70 4 gene was studied.The human β2m gene was injected into F1 fertilized eggs of C5 7BL 6 × Kunming mice and Kunming mice × Kunming mice by microinjection.The PCR products of born animals and their offspring , The positive genomic DNA samples were further identified by spot blot hybridization and Southern hybridization and the copy number was integrated and the expression of human β2m transgene in positive mice was detected by RT PCR.All the 6 primary pups and 7 their next generation mice F1) with human β2m gene.Among the 86 mice born from the post-microinjection gene posterior ova, 35 (7%) of the C5 7BL 6 × Kunming mice were hybridized, of which 4 were positive (114%), Kunming mice × Kunming mice There were 51 offspring, of which 2 were positive (39%), 20 were primary mice with human β2m gene × 20 Kunming mice, of which 7 were positive. The integrated transgenics were all single copies. The above positive mice do have transgene integration.The expression of human β2m transgenic mRNA was observed in the skin, colon, testes and spleen of positive mice.In the preparation of transgenic animals, F1 generation of C5 7BL 6 × Kunming mice was significantly better than that of Kunming mice × Kunming mice F1 generation and human HL A B2 70 4 gene, human β2m gene is not easy to integrate, the integration rate and the integrated copy number are low.The resulting human β2m transgenic mice can be difficult to pass on human β2m base to the next generation, and human HLA B2 70 4 transgenic mice mating to study its pathogenicity