猫视神经慢性损伤相关差异表达基因消减cDNA文库的构建

Construction of subtracted cDNA library for differentially expressed genes associated with chronic optic nerve injury

目的:构建猫视神经慢性损伤相关差异表达基因消减cDNA文库。方法:15只成年家猫随机分为正常对照组、视神经压迫4周组和视神经压迫8周组(n=5),压迫4周组和压迫8周组猫利用球囊植入法建立慢性视神经损伤模型。取各组动物视神经,TRIzol法提取总RNA.SMART技术合成cDNA,随后利用抑制消减杂交方法分离受压4周和8周视神经中差异表达基因的cDNA片段,将其与T载体进行T/A连接构建文库,将连接产物用氯化钙转化法转化大肠杆菌进行文库扩增和蓝白斑筛选。随机挑取300个白色克隆进行菌落PCR鉴定。结果:菌落PCR扩增显示每个文库80%的克隆中均有200～800 bp的插入片段。结论:成功构建猫视神经慢性损伤相关差异表达基因消减cDNA文库,为进一步筛选、克隆慢性视神经损伤相关差异表达基因奠定了基础。

Objective：To construct subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury in cats. Methods： Fifteen adult cats were randomly divided into 3 groups （n = 5） ： control group, 4-w compression group and 8-w compression group. The chronic optic nerve injury was produced by an inflatable balloon implanted under the optic chiasm. The total RNA was prepared from optic nerves of each group by TRIzol method. Double-stranded cDNA was produced by SMART PCR cDNA synthesis protocol. Suppression subtractive hybridization （SSH） was used to isolate the cDNA fragments of differentially expressed genes in the optic nerves after 4-w and 8-w compression. The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library, followed by amplification of the libraries through E. coli transformation with calcium chloride and screening of blue and white clones. Three hundred positive bacterial clones were randomly picked in each library and identified by colony PCR. Results： Analysis of the white clones by PCR showed that 80% clones contained 200-800 bp inserts in each library. Conclusion： Four subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury have been successfully constructed by SSH and T/A cloning techniques,which lays a solid foundation for screening and cloning specific differentially expressed genes associated with chronic optic nerve injury.