摘要
目的研究人类白细胞抗原(HLA)-B2704基因在转基因小鼠中的整合与表达。方法应用显微注射技术将HLA-B2704基因注入C57BL/6×昆明鼠和昆明鼠×昆明鼠F0代受精卵,出生动物进行聚合酶链反应(PCR)筛选及Southern杂交鉴定。阳性者进行反转录(RT)-PCR和流式细胞光度术检测其在mRNA和蛋白水平的表达。结果101只F0代仔鼠中,10只F0代仔鼠基因组中有HLA-B2704基因整合。其中昆明鼠×昆明鼠F0代3只,整合率为4.7%,C57BL/6×昆明鼠F0代7只,整合率为18.9%(字2检验,P<0.05)。阳性小鼠的皮肤、结肠、睾丸和脾脏组织中均有HLA-B2704基因的mRNA水平表达,其外周血淋巴细胞膜上HLA-B2704基因的蛋白表达为7.87%(直方图统计表,5%以上为阳性)。结论尽管受精卵的注射成活率差异无统计学意义,在注射浓度相同的情况下,含有C57BL/6基因背景的受精卵更易整合外源基因,而单纯昆明鼠的受精卵不易接受外源基因。C57BL/6×昆明鼠F0代比单纯昆明鼠F0代更适合于转基因动物的制备。HLA-B2704的重链分子与小鼠茁2微球蛋白结合的复合物不稳定,不易于在细胞表面表达。
Objective To study the integration and expression of HLA-B2704 gene in HLA-B2704 transgenic mice. Methods HLA-B2704 gene was introduced into fertilized eggs of C57BL/6×Kunming and Kunming×Kunming by microinjection. The founder mice were screened by PCR for integration of HLA-B2704 transgene and were then further confirmed by southern blot. RT-PCR and flow cytometry were carried out to detect the expression of HLA-B2704 gene at mRNA and protein level. Results Ten F0 hybrid mice carried HLA-B2704 gene in 101 F0 hybrid mice, 3 of 64 (4.7%) hybrid mice from Kunming×Kunming and 7 of 37 (18.9%) hybrid mice from C57BL/6×Kunming background carried HLA-B2704 gene. Statistical analysis showed that there was significant difference in the integration rate between the two groups (P<0.05). HLA-B2704 gene was expressed at mRNA level in the skin, colon, testicle and spleen tissue of the transgenic mice. The expression of HLA-B2704 gene on the cell membrane of their peripheral blood lymphocytes was 7.87% in HLA-B2704 transgenic mice at protein level. Conclusion Although the survival rate of fertilized eggs injected is not significantly different, the fertilized eggs of hybrid mice with C57BL/6 mice gene background are more easily integrated with exogenous gene, while the fertilized eggs of hybrid mice with Kunming mice gene background are integrated with difficult. Compared with Kunming×Kunming F0, C57BL/6×Kunming F0 is more suitable for production of transgenic mice. The complex of HLA-B27 heavy chain molecule combined with mouse β2 microglobulin is unstable, so it is difficult to express on the surface of the cell membrane.
出处
《中华风湿病学杂志》
CAS
CSCD
2005年第6期321-324,共4页
Chinese Journal of Rheumatology
基金
国家自然科学基金资助项目(3007064)
