Shkbp1等几种基因工程小鼠纯合品系的建立
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摘要
研究背景和目的:
当前,利用遗传工程方法制作人类疾病的基因工程小鼠已成为研究人类重大疾病(包括传染病、肿瘤、心血管疾病、代谢性疾病、老年性疾病、精神性疾病及遗传病等)的重要方法和手段。与普通实验小鼠相比,基因工程小鼠具有无法比拟的巨大优势,它能够模拟人类疾病在体内的自然发生过程,更有利于临床治疗研究和药物开发,同时它又能够将疾病的机理研究推进到分子水平,更有利于新的药物靶点的发现。
用基因工程小鼠与自发疾病小鼠模型杂交,来研究目的基因在疾病中的作用,是目前人类重大疾病功能基因研究中最先进的技术。为了更准确的研究上述基因在炎症、肿瘤等疾病中的功能,充分利用基因工程小鼠模型在生命科学研究的巨大优势,本实验室依据研究方向构建了4种基因工程小鼠,分别是Shkbp1基因剔除小鼠,SGTa基因剔除小鼠,pVA-hCD2-Nef基因剔除小鼠和胰岛过表达Slit2蛋白的Rip-hSlit2小鼠,并用遗传学方法获得具有稳定遗传背景的纯合子小鼠,希望通过与ApoE KO, Rip1-Tag2, ApcMin, TRAMP和MMTV-PvMT等疾病小鼠杂交,发现相关基因在炎症、肿瘤和血管疾病中的作用。
方法:
1基因工程小鼠的饲养
各品系基因工程小鼠饲养于SPF级环境下,根据需要进行配种,将哺乳期结束的幼鼠及时分笼,为纯合子小鼠的筛选做好准备。
2稳定遗传的基因工程小鼠品系的建立与纯合子的筛选采用经典遗传学中的孟德尔定律将转基因小鼠进行一定方式的交配,结合PCR方法检测目的基因。
3 Shkbp1基因对小鼠肿瘤生长的影响
利用Shkbp1基因剔除纯合子小鼠为实验组,野生型129小鼠作为对照组,建立B16黑色素瘤皮下移植瘤模型,通过测量肿瘤体积、肿瘤重量,初步探讨Shkbp1敲除后对小鼠肿瘤生长和转移的影响。
结果:
1通过129小鼠与F2代阳性小鼠测交,检测子代小鼠的基因型,筛选出1只雄性、3只雌性Shkbp1小鼠,其子代基因鉴定结果阳性率为100%;筛选出2只雄性、1只雌性SGTa小鼠,其子代基因鉴定结果阳性率为100%; pVA-hCD2-Nef小鼠,Rip-hSlit2小鼠筛选仍在进行中。
2 Shkbp1敲除对小鼠B16皮下移植瘤具有抑制趋势,但是统计结果没有显著性差异。
结论
1成功构建了Shkbp1基因剔除的纯合子小鼠,并初步验证了Shkbp1基因在肿瘤生长和转移中的作用
2成功构建了SGTa基因的纯合子小鼠模型。获得了F2代pVA-hCD2-Nef小鼠和F1代Rip-hSlit2小鼠。
Background & Objective
Currently, genetically engineered mice of human diseases model produced in genetic engineering have become an important method and means to study human diseases (including communicable diseases, cancer, cardiovascular diseases, metabolic diseases, age-related diseases, mental diseases and genetic diseases etc.). Compared with normal mice, genetically engineered mice have the un-compared dominance that can simulate the naturally occurring process of human disease in vivo and is more conducive to clinical research and pharmaprojects. It also can carry on studying the mechanism of the disease to the molecular level and is more conducive to the discovery of new drug targets.
Crossing genetically engineered mice with spontaneous disease mouse model to study the role of genes in disease, is the most advanced technology used to study the functional genes of human major diseases. In order to study the functions of refered genes in inflammation, cancer and other diseases more accurately, make full use of the great advantages of genetically engineered mouse models in life science research, four strains of genetically engineered mice were constructed by our laboratory, Shkbp1 knockout mice, SGTαknockout mouse, nef1 knockout mouse and overexpression of Slit2 protein Rip-hSlit2 pancreatic mice respectively. Genetic methods were used to obtain a stable genetic background homozygous mice, hopefully to find the roles of other genes related to inflammation, cancer and the role of vascular disease by crossing to ApoE KO, Rip1-Tag2, ApcMin, TRAMP and MMTV-PvMT mice and other diseased mice model.
Method
1 Breeding genetic engineering mice
Genetically engineered mice were housed in SPF-class environment, according to the need for breeding, allocating mice timely after lactation, to make preparation for the screening of homozygous mice.
2 The establishment of stable genetically engineered mice and screening of homozygous
Applied classic Mendelian laws in genetics to breed the transgenic mice, and combined with PCR to detect the target genes.
3 Shkbp1 gene on tumor growth
Making Shkbp1 homozygous knockout mice as the experimental group, wild-type 129 mice as the control group to establish subcutaneous B16 melanoma tumor model Measuring the tumor volume, tumor weight,to further investigate the effect of Shkbp1 knockout mice on tumor growth and metastasis.
Results
1 129 generation mice cross with F2 positive mice to detect the genotypes of their offspring. when one male and three female Shkbp1 mice have been selected, the offspring gene identification results was 100%; when two male and one female SGTαmice have been selected, the offspring gene identification results was 100%; when one male and one female pVA-hCD2-Nef in mice have been selected, the offspring gene identification result was 100 %. Rip-hSlit2 mouse filter is still in progress.
2 Shkbp1 in subcutaneous tumor and metastatic tumor mice model (the results are in statistics)
Conclusion
1 Shkbp1 homozygous knockout mice were successfully constructed, and it is verified the effect of Shkbp1 gene in tumor growth and preliminary metastasis.
2 SGTa homozygous knockout mice were successfully constructed. Generation F2 pVA-hCD2-Nef and generation F1 Rip-hSlit2 TG mice were constructed.
当前,利用遗传工程方法制作人类疾病的基因工程小鼠已成为研究人类重大疾病(包括传染病、肿瘤、心血管疾病、代谢性疾病、老年性疾病、精神性疾病及遗传病等)的重要方法和手段。与普通实验小鼠相比,基因工程小鼠具有无法比拟的巨大优势,它能够模拟人类疾病在体内的自然发生过程,更有利于临床治疗研究和药物开发,同时它又能够将疾病的机理研究推进到分子水平,更有利于新的药物靶点的发现。
用基因工程小鼠与自发疾病小鼠模型杂交,来研究目的基因在疾病中的作用,是目前人类重大疾病功能基因研究中最先进的技术。为了更准确的研究上述基因在炎症、肿瘤等疾病中的功能,充分利用基因工程小鼠模型在生命科学研究的巨大优势,本实验室依据研究方向构建了4种基因工程小鼠,分别是Shkbp1基因剔除小鼠,SGTa基因剔除小鼠,pVA-hCD2-Nef基因剔除小鼠和胰岛过表达Slit2蛋白的Rip-hSlit2小鼠,并用遗传学方法获得具有稳定遗传背景的纯合子小鼠,希望通过与ApoE KO, Rip1-Tag2, ApcMin, TRAMP和MMTV-PvMT等疾病小鼠杂交,发现相关基因在炎症、肿瘤和血管疾病中的作用。
方法:
1基因工程小鼠的饲养
各品系基因工程小鼠饲养于SPF级环境下,根据需要进行配种,将哺乳期结束的幼鼠及时分笼,为纯合子小鼠的筛选做好准备。
2稳定遗传的基因工程小鼠品系的建立与纯合子的筛选采用经典遗传学中的孟德尔定律将转基因小鼠进行一定方式的交配,结合PCR方法检测目的基因。
3 Shkbp1基因对小鼠肿瘤生长的影响
利用Shkbp1基因剔除纯合子小鼠为实验组,野生型129小鼠作为对照组,建立B16黑色素瘤皮下移植瘤模型,通过测量肿瘤体积、肿瘤重量,初步探讨Shkbp1敲除后对小鼠肿瘤生长和转移的影响。
结果:
1通过129小鼠与F2代阳性小鼠测交,检测子代小鼠的基因型,筛选出1只雄性、3只雌性Shkbp1小鼠,其子代基因鉴定结果阳性率为100%;筛选出2只雄性、1只雌性SGTa小鼠,其子代基因鉴定结果阳性率为100%; pVA-hCD2-Nef小鼠,Rip-hSlit2小鼠筛选仍在进行中。
2 Shkbp1敲除对小鼠B16皮下移植瘤具有抑制趋势,但是统计结果没有显著性差异。
结论
1成功构建了Shkbp1基因剔除的纯合子小鼠,并初步验证了Shkbp1基因在肿瘤生长和转移中的作用
2成功构建了SGTa基因的纯合子小鼠模型。获得了F2代pVA-hCD2-Nef小鼠和F1代Rip-hSlit2小鼠。
Background & Objective
Currently, genetically engineered mice of human diseases model produced in genetic engineering have become an important method and means to study human diseases (including communicable diseases, cancer, cardiovascular diseases, metabolic diseases, age-related diseases, mental diseases and genetic diseases etc.). Compared with normal mice, genetically engineered mice have the un-compared dominance that can simulate the naturally occurring process of human disease in vivo and is more conducive to clinical research and pharmaprojects. It also can carry on studying the mechanism of the disease to the molecular level and is more conducive to the discovery of new drug targets.
Crossing genetically engineered mice with spontaneous disease mouse model to study the role of genes in disease, is the most advanced technology used to study the functional genes of human major diseases. In order to study the functions of refered genes in inflammation, cancer and other diseases more accurately, make full use of the great advantages of genetically engineered mouse models in life science research, four strains of genetically engineered mice were constructed by our laboratory, Shkbp1 knockout mice, SGTαknockout mouse, nef1 knockout mouse and overexpression of Slit2 protein Rip-hSlit2 pancreatic mice respectively. Genetic methods were used to obtain a stable genetic background homozygous mice, hopefully to find the roles of other genes related to inflammation, cancer and the role of vascular disease by crossing to ApoE KO, Rip1-Tag2, ApcMin, TRAMP and MMTV-PvMT mice and other diseased mice model.
Method
1 Breeding genetic engineering mice
Genetically engineered mice were housed in SPF-class environment, according to the need for breeding, allocating mice timely after lactation, to make preparation for the screening of homozygous mice.
2 The establishment of stable genetically engineered mice and screening of homozygous
Applied classic Mendelian laws in genetics to breed the transgenic mice, and combined with PCR to detect the target genes.
3 Shkbp1 gene on tumor growth
Making Shkbp1 homozygous knockout mice as the experimental group, wild-type 129 mice as the control group to establish subcutaneous B16 melanoma tumor model Measuring the tumor volume, tumor weight,to further investigate the effect of Shkbp1 knockout mice on tumor growth and metastasis.
Results
1 129 generation mice cross with F2 positive mice to detect the genotypes of their offspring. when one male and three female Shkbp1 mice have been selected, the offspring gene identification results was 100%; when two male and one female SGTαmice have been selected, the offspring gene identification results was 100%; when one male and one female pVA-hCD2-Nef in mice have been selected, the offspring gene identification result was 100 %. Rip-hSlit2 mouse filter is still in progress.
2 Shkbp1 in subcutaneous tumor and metastatic tumor mice model (the results are in statistics)
Conclusion
1 Shkbp1 homozygous knockout mice were successfully constructed, and it is verified the effect of Shkbp1 gene in tumor growth and preliminary metastasis.
2 SGTa homozygous knockout mice were successfully constructed. Generation F2 pVA-hCD2-Nef and generation F1 Rip-hSlit2 TG mice were constructed.
引文
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