| mcprl gene (mouse cleft palate-related gene 1) is a novel gene (Gene bank access number:AY074887) .which was screened by PCR subtractive hybridization method, and highly expressed in mouse cleft palate tissues induced by retinoic acid. On the basis of sequence, mcprl was predicted encoding a protein comprised of 132 amino acids. But we still know few about the distribution and function of this gene/protein, one of the obscles is lack of MCPR1 protein and specific antibody, fau gene is also one of the different expressing genes. The purpose of present study was to detect the expression of these two genes, analysis their basic function and explore the relationship between genes and cleft palate. It will be helpful to prevent the common craniofacial congenital malformations if mechanism of the cleft palate developing is understood well. The studies include seven parts:1. This study was undertaken to detect the expression of the mcprl and fau gene in palatal process, brain, heart, liver, lung tissues etc. of normal C57BL/6N strain mouse. The mRNA was extracted respectively directly from these tissues and detectd the expression level by RT-PCR. The result showed that genes were expressed in many tissue of the normal mouse. So it was concluded that the extensive expression of genes in tissue was significant in development of normal tissue.2. The object of this study was to observe the expression of the mcprl and fau gene in cells derived from undifferentiating ectodermic palatal mesenchyme of C57BL/6N strain mouse. The expression of mcprl and fau gene in the cells was tested by in situhybridization. The result showed that genes were expressed in undifferentiating ectodermic palatal mesenchyme cells of the normal mouse. So it was concluded that there was the significance of mcprl and fau gene during developing of the palatal process in C57BL/6N strain mouse.3. The purpose of this investigation was to observe the expression of mcprl and fau gene in the formation of cleft palate of mice and to explore the relationship between genes and the formation of cleft palate. The expression of genes in embryonic palatine process tissues in the experimental group induced by retinoic acid and control group was detected by in situ hybridization. The data showed that the expressional level of genes was higher in the experimental group than in the control group. It suggested that the genes played an important role to maintain the growth and development of tissues of mice. The high level expression of such genes was related to the occurrence of cleft palate.4. The aim of this study was to amplify the mcprl gene and construct a high effective expressing vector containing mcprl cDNA. mcprl encoding region cDNA gene was amplified by PCR from the plasmid T-easy/ mcprl, then PCR product was inserted into a high effective fluorescent eukaryotic expressed vector pEGFP桸3. The positive recombinant was identified by PCR analysis, EcoRI and BamHI restriction analysis, and Sequence analysis. The result showed that a 400bp DNA fragment was amplified from the recombinant. Sequence analysis and restriction digest demonstrated that the mcprl gene was successfully inserted into pEGFP桸3 plasmid. So the fluorescent eukaryotic expressed vector pEGFP桸3/mcprl were successfully reconstructed. It would be useful for further studing the function of mcprl gene.5. In this study an attempt was made to express pEGFP-N3- mcpr1 fusion protein and to examine intercellular localization of MCPR1.pEGFP-N3- mcpr1 expression plasmid was transfected into COS7 by lipofectin reagent. After 48 hours, the location of the fusion protein in cells was observed under the fluorescent microscope. So it was concluded that MCPR1 localizes in the cytoplasm. The work provides the basis for study of the biochemical and physiological functions of mcprl.6. In this part, by stable transfection technique, the open reading fream of mcpr1 were sucessfully introduced into continous cell line COS7. After 1 month of G418 treatment, all survived cells and it' s decedents(for a...
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