Cloning, Screening, Location And Function Study Of Novel Cleft Palate Related Gene

Cleft palate is a one of the most common craniofacial congenital malformations. The incidence of the cleft palate is very high in Asia, special in china. In 1996 ~ 2000, the result of inspecting congenital malformations in chin a indicated that leading disease is cleft palate/lip. Nowadays the only therapy of this abnormity is to close the cleft by surgery. But the effect is not satisfaction and the patient is still handicapped on the function of pronounce and chew. It also affected the patient mental health. Cleft palate is a kind of polygenic inheritance. The etiology of cleft palate is complex, including multiple genetic and environmental factors. It will be helpful to prevent this common craniofacial congenital malformations if mechanism of the cleft palate developing is understand well. A subtractive hybridization method was performed in this work and the mRNA was isolated directly from palatal process both control group and RA treated group. The purpose of this study is to clone and screen different expressing genes, which were relating to cleft palate. The study was divided into three parts.1. This study was undertaken to observe the effects of MSX gene on cells derived from undifferentiating ectodennic palatal mesenchyme and discuss the significance of MSX gene during developing of the cleft palate in C57BL/6N strain mouse. The proliferation of the cells by MTT and the expression of MSX gene in the cells between RA treated and control group by ISH (In situ9hybridization, ISH). Result suggested the proliferation of the cells group was significantly inhibited and the level of MSX gene expression was significantly higher in RA treated. RA inhibited the proliferation of palatal mesenchymal cells through changing the expression of MSX gene. MSX gene was maybe the target gene of the RA during it induced the development of cleft palate.2. The purpose of this investigation was to construct a subtractive cDNA library of gene differentially expressed in palatal process between normal group and RA treated group C57BL/6N strain mouse. The mRNA was extracted respectively from palatal process in control and RA treated group C57BL/6N strain mouse on gestation da}' 12. The cDNA library was constructed by an improved technique based on PCR subtractive hybridization. Part of clones were randomly selected and identified with dot blot hybridization. Result: A subtractive cDNA library of cleft palate was constructed with a capacity of 7.6 X10 . We conclusion that construct a efficient and high capacity subtractive cDNA library of cleft palate will be a forceful tool for identifying mechanism of cleft palate in gene level. We continued to screen and clone the differently expression gene in palatal process between normal group and retinoic acid treated group mouse and explore the significance of the gene expression. 4 new genes were obtained. The others affecting on the proliferation and differentiation of cells during the developing cleft palate were ribosomal protein and fan gene. No.l new gene was identified as full-length cDNA sequence by northern blot and named MCPR-] gene (mouse cleft palate related gene-1). Analysis of MCPR-1 gene sequence showed that is a completed openreading frame encoding 132 amino acids.3. In this study an attempt was made to detect the expression of the MCPR-1 and fan gene in palatal process between experimental and control group. With RT-PCR we also explore two genes in brain, heart, liver, lung tissue etc. aged between Iday and 3 month C57BL/6N strain mouse. The data show that MCPR-1 gene was only expressed in palatal process in RA treated group. The10higher expression level of fau gene was significant in experimental group. Besides that MCPR-1 gene was not detected in all above tissues and fau expressed in brain, heart, liver, lung tissue etc in aged Iday mouse but only expressed in testine and kidney.