Expression Of Shh Gene In Limb Bud Of NIPBL^+/- Fetal Rats

Background and Objective:Cornelia de Lange Syndrome（CdLS）is a rare genetic disease associated with severe growth defects and multiple organ systems caused by genetic mutations.The mutation of these genes will lead to the disorder of gene expression,accompanied by a variety of severe bone abnormalities.It is mainly manifested as retardation of growth,cognitive impairment,upper limb deformity,hirsutism,special face and heart valve defects.It has seriously affected the healthy growth of newborns and children,and has caused a heavy burden on families and society.Among them,65%of the children were caused by three mutations of NIPBL,Smc1 and Smc3.The NIPBL gene mutation is the main cause of the autosomal dominant hereditary disease Cornelia de Lange Syndrome,and about 60%of the children are caused by the mutation of the NIPBL gene.In the development stage of fetal limb bud,NIPBL gene regulates the Shh gene located in the ZPA region of limb bud,and affects the expression of Shh gene.However,the interaction between two specific genes is not yet known.In this experiment,we used NIPBL-Loxp mice and Cre mice to establish NIPBL^+/-gene knockout mouse model,and on the basis of this model,we discussed the expression of Shh gene at different stages of fetal rat development.Methods:The background mice of NIPBL-Loxp mice and Cre mice were C57BL/6J,SPF grade,and they were purchased at Zhejiang University.All mice were bred in the experimental animal center of Shihezi University School of pharmacy.The experimental procedure follows the regulations of the management of the ethics committee of the animal experiment of Shihezi University.In this experiment,we first establish a mouse model of NIPBL-Loxp,and then use the NIPBL-Loxp mice and Cre mice to establish NIPBL^+/-gene knockout mouse model and NIPBL^+/-mice model as the parental mice,then select male NIPBL^+/-mice35g and female 30g.At 20:00 o’clock in the evening,the male and female mice were cooperated with 2:1.The female rats were checked at 8 o’clock next morning,and the female mice with negative suppositoryies were served as their embryonic E0.5 days at noon the next day.After crossing hybrid the NIPBL^+/-mice,the pregnant rats were removed on the E10,E11 and E12 days of the pregnant mice,and placed in the super clean table.The pregnant rats were killed by neck dislocations,and the fetuses were taken out under strict aseptic conditions.Fetal rats were taken out after using reverse transcriptase polymerase chain reaction（reverse transcription polymerase chain reaction respectively,RT-PCR）Technology identified 6NIPBL+/-fetal rat limb bud as the experimental group,and 6 NIPBL+/+fetal rat limb bud as the control group,then,separation of fetal rats limbs,and put it in liquid nitrogen and-80℃refrigerator spare;finally,the expression of Shh gene in the limb bud of the experimental group and the control group was detected by quantitative quantitative reverse transcription polymerase chain reaction（qRT-PCR）technology.Results:We use the NIPBL-Loxp mice and Cre mice to establish NIPBL^+/-gene knockout mouse model,NIPBL+/-mice were hybridized with each other and the fetal rats were identified by RT-PCR technology as experimental group and control group.Among them,the Shh gene in the limb buds of the experimental and control groups was expressed in E10,11,and 12 days and the Shh gene in the limb bud of the experimental group and the control group at E10 days ofΔCt value>E12 days ofΔCt value>E11days ofΔCt value（The higher the value of theΔCt,the lower the expression level of the Shh gene）.This indicates that the expression of Shh gene in the limb bud of the experimental group and the control group is higher than that of the control group at different days（E10,11,12 days）,and the difference is statistically significant（P<0.01）.In the same period of pregnancy（E10,11,12 days）,theΔCt value of the Shh gene in the experimental group was larger than that of the control group,indicating that the expression level of Shh gene in the limb bud of the experimental group was lower than that of the control group,this difference is also statistically significant（P<0.01）.Conclusion:In In the pregnant rats E10,E11 and E12 days,NIPBL^+/-gene knockout mouse fetal limb bud inhibits the expression of its own Shh gene,indicating that the expression level of Shh gene in the specific stage of fetal limb bud is helpful for people to better understand the pathogenesis of bone malformation in the Cornelia de Lange Syndrome.