摘要
目的 :通过SouthernBlot的方法鉴定PDGF D转基因鼠。方法 :显微注射Alpha MyHC PDGF D质粒后 ,产生的PDGF D转基因c5 7小鼠养至 4～ 6周。取 0 5～ 1 0cm的小鼠尾巴 ,蛋白酶K消化过夜 ,提取基因组DNA。PCR方法初步筛选 ,阳性鼠进行进一步的SouthernBlot验证。紫外分光光度法定量基因组DNA ,EcoRI酶切过夜 ,酶切产物经纯化后 ,电泳 ,转膜。PCR方法制备Alpha MyHC PDGF D载体上特异的hGHPolyA探针 ,然后由32 P dCTP标记探针 ,与转好的膜相互杂交 ,- 70摄氏度在X光片上曝光约 1个月 ,得到转基因鼠鉴定的结果。结果 :SouthernBlot显示 4笼共 2 0只PDGF
Objective To identify the PDGF-D transgenic c57 mice by Southern Blot. Methods： Transgenic mice were generated by microinjection of purified transgenic Alpha-MyHC-PDGF-D vector into the pronuclei of fertilized eggs. Transgenic mouse genomic DNA was isolated from mice tails. After identification with PCR, the positive mice were further identified with Southern Blot. 10 μg of the genomic DNA was digested with EcoRI, followed by fractionation through a 0.8 % agarose gel and transferred onto a nylon membrane. The hGH PolyA probe was produced by PCR. After the hybridization with a ^32P-labeled random-primed hGH PolyA probe, the membranes were subjected to autoradiography for about one month. Results： The results of Southern Blot indicated that there is only one positive PDGF-D transgenic mouse in these 20 mice.
出处
《中国分子心脏病学杂志》
CAS
2004年第2期84-88,共5页
Molecular Cardiology of China
