| In recent years,the quantity demand for mutton has been increased with the improvement of people’s living standard.The conservation and utilization of local mutton will effectively enhance the development level of sheep industry in China.Ujumqin Sheep belonging to Mongolian sheep is an important mutton breed in China.The mutant individuals with multiple vertebrae traits in Uzhumqin Sheep population can make significant increase of mutton production,which is of great significance to the development of mutton industry as well as the increase of herdsmen’s economic income in pastoral area.Phenogenetics study have demonstrated that multiple vertebrae traits is regulated by Homeobox gene(Hox)gene,which is caused by a few gene mutations and can be inherited steadily.It is predicted that Hoxc8 gene mainly regulates the formation of the thoracic vertebrae,and its mutation will lead to changes in the number of thoracic vertebrae development.However,these findings and predictions have not been fully validated by molecular biology.In this study,the CDS region of sheep Hoxc8 gene was cloned and its overexpression vector and knockout vector were constructed.The model of transgenic Adipose-Derived Stem Cells interfering Hoxc8 gene expression was established through screening monoclones after transfecting stem cells with different vectors.The changes of the genes and signaling pathways in different stem cell transcripts closely related to the changes of Hoxc8 gene expression was compared,so that to explore the effects of Hoxc8 gene on the regulation of stem cell metabolism,the change trend of Hoxc8 gene in the process of the stem cell differentiation into osteoblasts was further observed.Finally,through combining the data with the stem cell transcripts and osteogenic differentiation,the relationship between Hoxc8 gene and multiple vertebrae formation at the genomic level was analyzed.The results of this research will be benefit of further understanding the molecular mechanism of multiple vertebrae traits in Ujumqin Sheep.1.Acquisition of Hoxc8 gene overexpression and knockout stem cellsIn the proposed experiment,the CDS region of sheep Hoxc8 gene was cloned,and the ratio of reference sequence to NCBI was 100%.The CDS sequence was inserted into the polyclonal sites of mammalian eukaryotic expression vector,and Hoxc8 gene overexpression vector was constructed.The knockout vector of Hoxc8 gene was constructed using CRISPR-Cas9 technique according to the number of exons on the CDS of Hoxc8 gene.The construction of the two vectors provided plasmid materials for transgenic cells acquisition in the next step.Adipose-Derived Stem Cells were transfected with Hoxc8 gene overexpression vector,then were selected by G148 screening method and limited dilution method respectively to screen the positive cells.Based on the comparison of the cell viability and apoptosis,there was no significant difference between the two selection methods.The proliferation activity of the transgenic cells obtained by limited dilution method was higher than that of by G418 screening method,and their cell growth cycle was shorter,therefore the overexpression cell lines were obtained by finite dilution method in this research.After Hoxc8 gene knockout vector was transfected into the stem cells,the knockout cell lines were obtained by limited dilution method and single cloning method respectively.The results showed that the positive cells collected by single cloning method were in shorter cell growth cycle.The mRNA expression levels and protein expression levels of Hoxc8 gene in overexpression and knockout cell lines were detected respectively.The results showed that the mRNA expression level of Hoxc8 gene in overexpression cell lines was 7 times higher than that of in control group,and there was no expression of Hoxc8 protein in knockout cell lines.In the current study,we obtained transgenic stem cell lines with high and low Hoxc8 gene expression,providing a reliable cell model for the future study of Hoxc8 family metabolism regulation in stem cells.2.Analysis of gene differences expression between Hoxc8 overexpression and knockout stem cellsHigh throughput transcriptome sequencing was performed on Hoxc8 overexpresseion cell lines,Hoxc8 knockout cell lines and control cells,a total of 142 756 374 Raw Reads were obtained.After data filtering,totally 137 846 948 Clean Reads were obtained,and 93.3% data of the total sequence in samples had a base number greater than 30(error rate less than 0.1%).The results of transcriptome sequencing of Hoxc8 overexpression cells and control cells showed that 2 271 differentially expressed genes were up-regulated and 5 690 differentially expressed genes were down-regulated(P < 0.05).The results of transcriptome sequencing of Hoxc8 knockout cells and control cells presented that 2 563 differentially expressed genes were up-regulated,and 5413 differentially expressed genes was down regulated(P <0.05).These differentially expressed genes were analyzed using GO function analysis.Among the molecular functions of genes,the most differentially expressed genes were found in three modules,organelle,cell part and cellular process,respectively.Signal pathways in KEGG was enriched and analyzed by hypergeometric test.The result showed that 16 differentially expressed genes were significantly enriched in two groups,including actin cytoskeleton regulation and MAPK pathway.The transcriptome results provide materials and data for further exploring the Hoxc8 gene interacting gene network in stem cells.3.Effect of Hoxc8 gene on the process of osteogenic induction of stem cellsThe process of osteogenic induction of Adipose-Derived Stem Cells showed that osteoblasts differentiated and formed at 17-21 days with the continuous high expression of Osteocalcin(OCN),alizarin red and alkaline phosphatase staining were positive.The expression of Hoxc8 gene increased significantly during the 9-12 days of induction period,and then decreased to the level before induction in the late induction period.Combined with previous transcriptome data,it was found that the changes of Hoxc8 gene were closely related to the signal factors in MAPK signaling pathway associated with Segmentation Clock.The results provided experimental evidence for exploring the relationship between Hoxc8 gene and the development of Ujumqin Sheep body segments.4.Analysis of genome resequencing data of Ujumqin Sheep to find the difference of Hoxc8 gene at genomic levelThe current work compared the genomic libraries of multiple vertebrae individuals and non-multiple vertebrae individuals of Ujumqin Sheep,as well as analyzed the difference regions between groups,and the difference annotation regions were not found in Hoxc8 gene at the genomic level.However,combined with the above transcriptome sequencing analysis,two genes with different coding regions in the Indel differential region,MDFI and EGFL8,were found to relate to the regulatory relationship with Hoxc8 gene changes. |
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