| Mouse is one of the most important model animals.Its genome sequence information has been analyzed by mouse genomes project(MGP),and 99% of the genes are homologous to human genes.The physiological structure and metabolic processes of mice are similar to humans.It is the most ideal model for studying human complex traits.In mouse genetics,the strategies for quantitative trait loci(QTL)localization are mainly divided into linkage analysis and association study.However,the traditional inbred mouse subspecies has a single source and lack of genetic diversity and it is difficult to rapidly locate functional genes related to the complex traits.Therefore,there is an urgent need to develop new mouse resources to meet our research needs.The genome of chromosomal subsititution mice is characterized by the fact that it only contains one donor mouse chromosome and genetic background other than this chromosome of the recipient mouse.The study focuses on a chromosome with a uniform and known genome background,and the QTL mapping effect is more Stable and easy to detect.Over the years,our research team has collected wild mice from 25 different regions of China,using wild mice as donors and C57BL/6J(B6)mouse strains as recipients,and continuously backcrossing through 6-8 generations and self-crossing through 3-5 generations of brothers and sisters.We successfully constructed the chromosome 1 substitution lines(C1SLs).The chromosomes of this population were replaced by B6 mice except that chromosome 1 was derived from wild mice.Through blood biochemical analysis,we found that there were significant differences in the lipid and glucose levels of C1 SLs from different regions,and mice with different degrees of hind limb defect were found in the strains derived from Guangxi Daxin.In order to identify genes related to blood lipid and glucose regulation and limb development defects in C1 SLs population,this paper mainly carried out the following two parts of research.Part I: Screening candidate genes for dyslipidemia in C1 SLs using liver expression profiling.Mouse chromosome 1 contains a large number of QTLs associated with lipid metabolism,but many lipid metabolism genes are still not localized.We constructed a wild mouse chromosome 1 substitution lines(C1SLs)in which all chromosomes were identical to B6 mice except for chromosome 1.Therefore,the difference in phenotype between C1 SLs and B6 can be inferred from the difference of chromosome 1.We tested blood lipids and blood glucose levels in 13 C1 SLs mice(chromosome 1 from Shanghai Jiading,Zhejiang Hangzhou,Shanghai Chongming,Shandong Linyi,Yunnan Kunming,Shandong Zaozhuang 1,Shandong Zaozhuang 2,Henan Sanmenxia,Zhejiang Balidian,Shanghai Songjiang,Jiangsu Tianwang,Guangxi Daxin and Shanghai Yangpu YP)and B6 mice,and found there is a significant difference of total cholesterol(CHOL),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C)and blood glucose(GLU)levels between C1 SLs and B6 mice.Compared with C1 SLs mice,B6 mice showed relatively low levels of GLU,CHOL,HDL-C and LDL-C,as well as relatively high levels of TG,and different C1 SLs also showed diverse differences in blood lipids and blood glucose levels.Using the weighted gene co-expression network analysis(WGCNA)method,we identified eight gene modules related to blood lipids and blood glucose levels by detecting the expression levels of the liver transcriptome gene in each mouse strain.Among these modules,module M19(83 genes)was significantly associated with all five traits.Modules M7(491 genes),M8(311 genes)and M12(247 genes)were significantly associated with glucose levels.Modules M1(930 genes),M14(99 genes),M20(389 genes)and M21(117 genes)were significantly associated with TG.Further joint human genome-wide association analysis revealed 48 candidate genes that regulate blood lipids or blood glucose.In addition,we found 38 genes related to blood lipids and blood glucose metabolism on chromosome 1,13 of which were reported as functional genes in the mouse model,and 25 were newly discovered lipid and glucose regulation candidate genes.These results indicate that C1 SLs are ideal mouse models for studying complex traits.Part II: Localization,cloning and functional study of genes related to limb developmental defects in mice.During the constructing wild mice chromosome 1 substitution in 25 different regions of China,we found that mice with different degrees of hind limb defect of the mouse substitution line derived from Guangxi Daxin.With genome-wide sequencing,we successfully mapped the trait-related segments to the 68.3Mb-73.3Mb on chromosome 8.Through the functional annotation of genetic variation in candidate segments of limb-deficient mice,we found that 27 genes had SNPs that significantly affected gene function.By screening the embryo hindlimb expression database,we found that 15 genes in the candidate segment were expressed in the embryo.By screening the mouse SNP database,it was found that 9 genes SNPs were present in common inbred mice,leaving 6 genes as candidate genes for subsequent studies.By detecting the limb bud expression profile of limb-deficient mice and normal control mice through RNA-seq technology,we found that the expression level of the candidate gene Pbx4 was significantly decreased in limb-deficient mice.We speculated the Pbx4 gene as the limb development-related gene to conduct functional studies.The Pbx4 gene is a member of the pre-B cell leukemia transcription factor family.It encodes a protein containing a homeobox DNA binding domain and serves as a key cofactor for the HOX protein,playing a key role in both embryonic development and cell differentiation.Using the mouse mesenchymal cell C3H/10T1/2 cell model,the Pbx4 gene was overexpressed to detect changes in limb development-related gene expression.When the Pbx4 gene was overexpressed 33.7-fold,the Hoxc4,Hoxc5,Hoxd8,Fgf4,Shh,Tbx5 and Wnt5 a genes increased significantly(5-30 fold),while Hoxa7,Hoxb2,Hoxb8,Hoxc10,Hoxd10,Fgf10,Pitx1,and Wnt4 genes possessed s slight increase in expression(2-5 times).This indicates that the Pbx4 gene plays a significant role in promoting the expression of limb development-related genes in C3H/10T1/2 cells.Construction of a CRISPR/Cas9-based Pbx4 knockout mouse model combined with N-methyl-N-nitro-N-methylnitrosoguanidine(MNNG)induction.The experimental group Pbx4(+/-)self-crossing pregnant mice and the control group B6 pregnant mice were intraperitoneally injected with 200mg/kg dose of MNNG,and the ratio of limb defects between wild type mice and knockout mice was found to be significantly different.Half of the Pbx4 heterozygous knockout mice and homozygous knockout mice showed limb developmental defects,while no wild-type mice showed limb-deficient,indicating that the Pbx4 gene is involved in embryonic limb development.Classical inbred mice face challenges in the genetic study of complex traits.This paper uses wild mice chromosome 1 substitution lines to explore genes related to blood lipid and glucose regulation and limb development.The results showed that there was abundant genetic and phenotypic diversity in wild mice chromosome 1 substitution lines,which provided a new mouse resource for genetic research.It is an ideal animal model for studying complex traits and locating related functional genes. |
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