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目的通过筛选高温致畸金黄地鼠胚胎神经管cDNA文库,克隆高温致畸相关基因CDK109。方法利用本室构建的高温致畸胚胎神经管cDNA文库,采用噬菌斑原位杂交方法,用寡核苷酸探针筛选cDNA文库,挑取阳性噬菌斑,将其转化为质粒,再通过限制性酶切鉴定阳性克隆并测序,将该基因标记探针与孕8.5 d高温致畸胚胎神经管组织和正常对照胚胎神经管组织总RNA进行NORTHERN杂交,以便确认该基因在高温致神经管畸形中的异常表达。结果成功地从高温致畸金黄地鼠胚胎神经管cDNA文库中筛选出高温致畸相关基因CDK109全长,其在高温致畸组的表达明显高于对照组。结论 CDK109在神经管中的高表达与高温致神经管畸形密切相关。
OBJECTIVE: To clone the cDNA library of embryonic neural tube of high temperature teratogenicity golden hamster and clone high temperature teratogenicity related gene CDK109. Methods The neural tube cDNA library of hyperthermic teratogenic embryos was constructed. The cDNA library was screened by oligonucleotide probe using plaque in situ hybridization. Positive plaques were picked and transformed into plasmids. Positive clones were identified by restriction enzyme digestion and sequencing. Northern blotting of the gene-labeled probe with the total RNA of embryonic neural tube tissues of embryonic day 8.5 embryos and normal control embryos was performed to confirm that the gene was expressed in neural tube defects induced by hyperthermia Abnormal expression. Results The full-length cDNA of CDK109, a high temperature teratogenicity-related gene, was successfully screened from the neural tube cDNA library of high temperature tempering golden hamster embryos. The expression of CDK109 in hyperthermia teratogenic group was significantly higher than that of the control group. Conclusion The high expression of CDK109 in neural tube is closely related to neural tube defects caused by hyperthermia.