SIc33a1错义突变S113R导致小鼠纯合子胚胎早期致死

发布时间：2019-06-26 21:26

【摘要】：[研究背景] SLC33A1(Solute carrier family33, member1)是在研究神经节苷脂O-乙酰化的过程中被发现的一个基因,位于人类染色体3q25,因为其可以将细胞质中的乙酰辅酶A转运至高尔基体,为神经节苷脂O-乙酰化提供乙酰基,所以又名AT-1(the acetyl-Co A transporter1)。研究发现很多内质网蛋白可以被SLC33A1调节,包括β位淀粉样前体蛋白裂解酶1(β-site APP cleaving enzyme1, BACE1)。淀粉样蛋白前体(amyloid protein precursor, APP)和低密度脂蛋白受体(low density lipoprotein receptor, LDLR)等,这些蛋白质在翻译修饰后对于膜结构的组装和分泌蛋白的运输有重要作用。如果SLC33A1的表达异常,则会引起一系列神经系统相关的疾病,如散发性肌萎缩侧索硬化(amyotrophic lateral sclerosis, ALS)、晚发性阿尔茨海默病(Alzheimer's disease, AD)等。本实验室2008年在山东青岛地区发现了一个遗传性痉挛性截瘫(hereditary spastic paraplegia, HSP)大家系,呈常染色体显性遗传。采用全基因组扫查方法和定位候选策略确定人类SLC33A1基因是该家系的致病基因,患者均携带S113R错义突变,家系中的正常个体和群体中未检测到该突变。斑马鱼实验显示,抑制斑马鱼slc33al基因表达影响运动神经元轴突生长,这种异常能被人类野生型SLC33A1基因拯救而不能被突变型拯救。由以上研究可知,SLC33A1对于维持神经元『正常功能非常重要,但SLC33A1表达异常导致HSP的作用机制尚不明确,因此,有必要深入研究和探讨SLC33A1蛋白的功能和作用机制。 [研究目的] 通过构建slc33al突变基因敲入小鼠模型,检测SLC33A1突变对小鼠表型的影响,以探讨SLC33A1的功能。 [研究方法] 构建slc33al突变基因敲入小鼠模型,并通过Slc33al突变基因敲入小鼠的杂合子与杂合子交配,检测子代小鼠基因型是否符合预期分离比例。如果不能得到纯合子新生小鼠,提示纯合子小鼠在胚胎期致死,则通过解剖杂合子与杂合子交配的孕鼠分离得到不同胚胎时期的胎鼠(E3.5、E7.5、E18.5),利用单纯PCR扩增法和PCR-RFLP法对胎鼠进行基因型鉴定。根据以上各个发育时期胚胎的基因型鉴定和分型结果,确定纯合子小鼠的致死时间。 [研究结果] 成功构建slc33a1突变基因敲入小鼠模型。对杂合子小鼠交配子代基因型分析结果显示,可以获得预期比例的杂合子,但不能得到纯合子小鼠,提示纯合子小鼠胚胎期致死。通过解剖与杂合子雄鼠交配的杂合子孕鼠,得到的E7.5和E18.5胚胎均无纯合子,E3.5胚胎有少量纯合子存活。 [结论] SLC33A1基因突变导致纯合子小鼠胚胎早期致死,SLC33A1对早期胚胎发育有非常重要的作用。
[Abstract]:[background] SLC33A1 (Solute carrier family33, member1) is a gene found in the study of gangliosides O-acetylation, which is located on human chromosome 3q25. because it can transport acetylcoenzyme A from cytoplasm to Gore matrix and provide acetylgroup for gangliosides O-acetylation, it is also known as AT-1 (the acetyl-Co A transporter1). It has been found that many endoplasmic reticulum proteins can be regulated by SLC33A1, including 尾-site APP cleaving enzyme1, BACE1. Starch protein precursor (amyloid protein precursor, APP) and low density lipoprotein receptor (low density lipoprotein receptor, LDLR), these proteins play an important role in the assembly of membrane structure and the transport of secretory proteins after translation modification. If the expression of SLC33A1 is abnormal, it will cause a series of nervous system related diseases, such as sporadic amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), late onset Alzheimer's disease (Alzheimer's disease, AD) and so on. In 2008, our laboratory found a genetic spastic paraplegia (hereditary spastic paraplegia, HSP) family in Qingdao, Shandong Province, showing autosomal dominant inheritance. The whole genome scanning method and localization candidate strategy were used to determine that human SLC33A1 gene was the pathogenic gene of the pedigree. All the patients carried S113R missense mutation, and the mutation was not detected in normal individuals and populations in the family. Zebrafish experiments showed that inhibition of slc33al gene expression in zebrafish affected axonal growth of motor neurons, which could be saved by human wild type SLC33A1 gene but not by mutant. From the above studies, it can be seen that SLC33A1 is very important to maintain the "normal function of neurons, but the mechanism of HSP caused by abnormal expression of SLC33A1 is not clear. Therefore, it is necessary to further study and explore the function and mechanism of SLC33A1 protein. [objective] to detect the effect of SLC33A1 mutation on the phenotype of mice by constructing the mouse model of slc33al mutant gene knockout in order to explore the function of SLC33A1. [methods] the mouse model of slc33al mutant gene knockout was established, and the heterozygote and heterozygote of Slc33al mutant gene knockout mice were mated to detect whether the genotype of the offspring mice was in accordance with the expected segregation ratio. If the homozygotic neonatal mice could not be obtained, suggesting that the homozygotic mice died in the embryonic stage, the fetal mice (E3.5, E7.5, E18.5) were isolated from the pregnant mice mated with heterozygote and dissected. The genotyping of the fetal mice was identified by simple PCR amplification and PCR-RFLP. According to the genotypic identification and typing of embryos at each developmental stage, the lethal time of homozygous mice was determined. [results] the mouse model of slc33a1 mutant gene knockout was successfully constructed. The results of genotypic analysis of gametophyte generation in heterozygous mice showed that the expected proportion of heterozygotes could be obtained, but the homozygotic mice could not be obtained, suggesting that the embryo death of homozygous mice. By dissecting the heterozygotic pregnant mice mated with heterozygous male mice, the E7.5 and E18.5 embryos had no homozygote, and a small amount of homozygote survived in E3.5 embryos. [conclusion] SLC33A1 gene mutation leads to early embryo death in homozygous mice. SLC33A1 plays a very important role in early embryonic development.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R363

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