Apr3条件性基因敲除小鼠模型的建立及初步表型研究

Establishment of the mouse model of conditional knockout of Apr3 and preliminary study on the phenotypes

目的建立凋亡相关蛋白3(Apr3)条件性基因敲除小鼠模型,并初步研究其表型。方法设计基因敲除方案,获得Apr3flox(+/-)嵌合体小鼠,繁殖获得Apr3(-/+)小鼠后,再自交得到Apr3基因全身敲除Apr3(-/-)纯合小鼠和对照野生型小鼠。提取小鼠鼠尾组织基因组DNA,PCR扩增野生型和敲除基因片段,在DNA水平判断小鼠的基因型;提取小鼠肝脏RNA和蛋白,利用RT-PCR和Western blotting技术检验Apr3基因的表达;观察基因敲除小鼠的遗传性状、体质量变化趋势以及可能影响生长发育和代谢的指标如血糖、血脂、肝功能;对小鼠溶酶体富集的主要脏器行苏木精-伊红染色(H-E染色)观察。结果成功建立Apr3基因敲除小鼠模型,其中Apr3(-/-)小鼠存活并且可育,在2月龄时体质量落后于野生型小鼠。肝功能检测结果显示,2月龄Apr3(-/-)小鼠谷草转氨酶(AST)较野生型小鼠升高(P<0.05);H-E染色结果显示,肝组织出现明显肝细胞水肿样变性和汇管区炎症细胞浸润。结论初步建立了Apr3条件性基因敲除小鼠模型;Apr3基因敲除引起2月龄小鼠体质量落后、肝脏组织形态学异常和AST升高。

Objective To establish the mouse model of conditional knockout of apoptosis related protein 3 （Apt3） and preliminary explore the phenotypes. Methods The knockout plan was designed andApr31 -j ＋1 chimeric mice were obtained and bred to get Apr3（ -/ -） mice. The Apr3（ -/ -） homozygous mice with complete knockout of Apr3 were obtained by inbreeding and wild type control mice. DNAs of genome of mouse tail tissues were extracted. Wild type and knockout gene fragments were amplified by PCR and the phenotypes of mice were determined at DNA level. RNAs and proteins of mouse livers were extracted and the expression of Apr3 gene was detected by RT-PCR and Western blotting. The genetic traits and trend of body weight change of Apr3 knockout mice and indexes that might affect the growth, development, and metabolism, such as blood glucose, blood lipid, and liver function, were observed. Major lysosomal accumulated organs were hematoxylin-eosin stained and observed. Results The mouse model of Apr3 knockout was successfully established. Apr3（ -/ -） mice were survived and fertile, but the body weight of two-month-old Apr3（ -/ -） mice was smaller than that of wild type mice. Results of the liver function test showed that the aspartate aminotransferase （AST） level of two- month-old Apt3（ -/ -） mice was higher than that of wild type mice （P〈0.05）. Results of H-E staining indicated significant degeneration and edema of liver cells and periportal inflammatory cell infdtration. Conclusion The mouse model of conditional knockout of Apr3 is established. The knockout of Apt3 causes low body weight, abnormal morphology of liver tissue, and elevated AST of two-month-old mice.