牛睫毛毛囊生长期毛乳头细胞cDNA表达文库的构建

为构建高质量的牛睫毛毛囊生长期毛乳头细胞cDNA表达文库,以新生荷斯坦牛上眼睑为组织材料,显微解剖分离出高纯度的生长期牛睫毛乳头,Trizol一步法提取总RNA后纯化mRNA;用ZAP Ex-press cDNA Synthesis Kit合成双链cDNA,进行末端修饰后接入ZAP表达载体。经GigapackⅢGold Pack-aging extract体外包装,转染XL1-Blue MRF′宿主菌,形成初级文库后进一步扩增,形成稳定的cDNA文库,文库库容量为3.3×107pfu/mL,重组率达95%。为进一步筛选、克隆生长期睫毛毛乳头特异性表达的基因、阐明睫毛毛囊发育的基因调控之分子机制奠定了研究基础。

Construction of Dermal Papilla cDNA Library of Bovine Anagen Eyelash Follicle

To construct high quality dermal papilla cDNA library of the bovine anageney elash follicle, the upper eyelids of neonatal Holstein cattle were used as experimental material,and the high purity dermal papilla was isolated under microscopy.The total RNA was extracted by Trizol one-step method and the mRNA was pured.The double-strand DNA was synthesized with ZAP Express cDNA Synthesis Kit,dsDNA was ligated with EcoR adapter and phosphorylated,then was digested by Xho.The cDNA fragments were ligated with the ZAP Express vector,and the ligated cDNA were packed and incubated with XL1-Blue MRF to construct primary library.The primary library was amplified to make astable library,the capacity of this library is 3.3×10⁷fu/mL and the recombination rate is 95%.These results indicate that a high quality cDNA library has been constructed.This library provides reaserch basis for screening and cloning of the gene expressed by the anagen eyelash dermal papilla,elucidating the molecular control of eyelash follicle morpho genesis as well.