Homozygous Embryos With Slc33a1Missense Mutation S113R Showed Early Developmental Arrest

[Background]SLC33A1(Solute carrier family33, member1) is a gene found in the study of ganglioside O-acetylation, and is located in human chromosome3q25. Because it can transfer the acetyl coenzyme A in the cytoplasm to the Golgi apparatus, to provide ganglioside O-acetylation with acetyl groups, it is also known as the AT-1(the acetyl CoA-from1). Studies have found that a lot of endoplasmic reticulum proteins can be regulated by SLC33A1, including (3-site APP cleaving enzyme1(BACE1)、 amyloid protein precursor(APP)and low density lipoprotein receptor(LDLR), etc. After translation and modification, these proteins play important roles for the assembling of membrane structure and transportation of secreted proteins. If the expression of SLC33Alis abnormal, it will cause a series of nervous system diseases, such as sporadic amyotrophic lateral sclerosis (ALS), late-onset Alzheimer’s disease (AD), etc.Previously, we found a family with hereditary spastic paraplegia (HSP) in Qingdao region of Shandong in2006, which is autosomal dominant inheritance. By genome-wide scan approach and positional candidate strategies, we determined human SLC33A1genes as the disease-causing gene of this family. All patients carry S113R missense mutation, and the mutation was not detected in normal individuals and controls. Zebrafish experiments have shown that inhibition of zebrafish slc33al gene expression results in the defects in motor neuron axon, and this abnormity can be rescued by human wild type SCL33A1, but not by mutant gene. These results indicated that SLC33A1is very important for neurons to maintain normal function. However, the mechanism of SLC33A1mutation causing HSP is not clear.Therefore, it is necessary to take in-depth study and exploration of the function and mechanism of SLC33A1protein.[Objective]By generating slc33al mutation knock-in mouse model, test the effects of SCL33A1mutation on phenotype of mice, and discuss the function of the SLC33A1.[Methods]The target vector was constructed, in which human mutation S113R was introduced into mouse slc33al gene, and a neo gene flanked by two loxp sites were inserted into the vector for ES cell selection. Heterozygous mice were crossed to wild type or heterozygous mice, and pups or embryos were obtained and analyzed for genotypes and phenotypes. Genotyping was performed by PCR and PCR-RFLP.[Results]We generated a mouse model with slc33al S113R mutation. When heterozygous mice were crossed to wild type mice, heterozygotes were recovered at a ratio expected of Mendelian inheritance. However, when heterozygous mice were crossed to heterozygotes, no homozygous mutant mice were present in the weaned pup, suggesting that homozygous mutant conceptuses can not develop to term or die before weaning. To determine the exact time point when homozygous conceptuses die, timed mating was performed and embryos at E18.5, E7.5, and E3.5were dissected. No homozygous mutant conceptuses were recovered at E7.5and E18.5, while several homozygotes were recovered at E3.5, suggesting that the homozygous embryos arrested at early development. [Conclusion]SLC33A1gene S113R mutation is a disease-causing mutation, and SLC33A1has a very important role in early embryonic development.