Homologous recombination between repeated chromosomal sequences in mouse cells.
Homologous recombination between repeated chromosomal sequences in mouse cells.
复制标题
小鼠细胞中重复染色体序列之间的同源重组。
DOI：
10.1101/sqb.1984.049.01.021
复制
发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Letsou,A
中科院分区：
文献类型：
--
作者：
Liskay,RM;Stachelek,JL;Letsou,A
材料和方法质粒构建。单纯疱疹病毒1型胸苷ki-183 nase基因（HSV-1 tk）的XhoI接头插入突变是大卫·齐普瑟博士（加州大学，拉霍亚）和杰西·郭博士（萨斯喀彻温大学，萨斯卡通）的慷慨馈赠。我们选择质粒载体pSV 2 neo（Southern和贝格1982）用于将HSV-1 tk突变基因插入tk小鼠L细胞。通过标准方法改变pSV 2neo上的各种限制性内切酶位点，以便于接受突变体HSV-I tk片段并提供用于线性化的独特位点。EcoRI位点改为ClaI，HindIII位点被删除，并在PvuI位点上创建一个新的HindIII位点。这个新产生的HindIII位点以及独特的BamHI位点是HSV-1 tk突变基因的靶标。从琼脂糖（International Biotechnologies，Inc. [IBI]）或丙烯酰胺（Bethesda Research Laboratories [BRL]）凝胶。在插入各自的位点之前，所有片段都用供应商（新英格兰Biolabs）推荐的HindIII或BamHI合成接头连接。这些研究中使用的质粒（见图1）携带含有HSV-1 tk突变基因26号（tk 26）的1.8kb HindIII片段和含有HSV-1 tk突变基因8号（tk 8）的2.4kb BamHI片段。根据瓦格纳等人的编号系统，XhoI接头插入突变相距485 bp，并定位在HSV-1 tk编码区的第735位（tk 26）和第1220位（tk 8）。（1981年）。
MATERIALS AND METHODSPlasmid construction. XhoI linker insertion mutations of the Herpes simplex virus type 1 thymidine ki-183 nase gene (HSV-1 tk) were a generous gift from Drs. David Zipser (Univ. of California, La Jolla) and Jesse Kwoh (Univ. of Saskatchewan, Saskatoon). We chose the plasmid vector pSV2neo (Southern and Berg 1982) for insertion of the HSV-I tk mutant genes into tkmouse L cells. Various restriction endonuclease sites on pSV2neo were altered by standard procedures to facilitate acceptance of the mutant HSV-I tk fragments and to provide a unique site for linearization. The EcoRI site was changed to ClaI, the HindlII site was removed, and a new HindlII site was created at the PvuI site. This newly created HindlII site, as well as a unique BamHI site, are the targets for the HSV-1 tk mutant genes. All DNA fragments used for cloning were recovered from agarose (International Biotechnologies, Inc.[IBI]) or acrylamide (Bethesda Research Laboratories [BRL]) gels by electroelution. All fragments were linked with either HindlII or BamHI synthetic linkers as recommended by the supplier (New England Biolabs) prior to insertion into the respective sites. The plasmid (see Fig. 1) used in these studies carries a 1.8-kb HindlII fragment containing HSV-I tk mutant gene number 26 (tk26) and a 2.4-kb BamHI fragment containing HSV-I tk mutant gene number 8 (tk8). The XhoI linker insertion mutations are 485 bp apart and map in the coding region of HSV-1 tk at positions 735 (tk26) and 1220 (tk8), according to the numbering system of Wagner et al.(1981).