Integration and activity of mammary tumor virus genes: regulation by hormone receptors and chromosomal position.
Integration and activity of mammary tumor virus genes: regulation by hormone receptors and chromosomal position.
复制标题
乳腺肿瘤病毒基因的整合和活性：激素受体和染色体位置的调节。
DOI：
10.1101/sqb.1981.045.01.086
复制
发表时间：
1981
期刊：
影响因子：
--
通讯作者：
Feinstein,SC
中科院分区：
文献类型：
--
作者：
Yamamoto,KR;Chandler,VL;Ross,SR;Ucker,DS;Ring,JC;Feinstein,SC
材料和方法DNA转化。根据GK Sim和R.阿克塞尔（哥伦比亚大学内科与外科学院，纽约）。含有MTV DNA插入物的重组噬菌体（A7. a）在CsCl中结合，并用无菌10 mM MgCl 2、10 mM HEPES（pH 7.0）和100 mM NaCl透析;用相同的缓冲液将透析的噬菌体稀释至1·10 11至1·10 12的滴度，然后调节至0.8M NaCl、30 mM MgCl 2、20 mM CaCl 2、8 mM HEPES（pH 7.0）。等体积的无菌60%聚乙二醇（Fisher PEG 6000），在转化前48小时，将细胞接种在2.5 × 105/100-mm培养皿中，并重新加入生长培养基（含有10%马血清的Dulbecco改良Eagle培养基[DMEM]）20-24小时。从板中吸出培养基，用磷酸盐缓冲盐水冲洗细胞;将1.1 ml噬菌体悬浮液与1 ml预热的无血清DMEM一起加入细胞中。在37 ° C孵育15分钟后，加入另外2 ml不含血清的DMEM。在37 ℃下继续孵育60分钟，然后加入10 ml生长培养基。4小时后和20小时后，更换培养基
MATERIALS AND METHODSDNA transformation. Transformation was carried out according to an unpublished procedure kindly provided by GK Sim and R. Axel (Columbia University, College of Physicians & Surgeons, New York). Recombinant phage containing an MTV DNA insert (A7. la) were banded in CsC1 and dialyzed against sterile 10 mM MgC12, 10 mM HEPES (pH 7.0), and 100 mM NaCl; the dialyzed phage were diluted with the same buffer to a titer of I• l0 II to 1• l012, then adjusted to 0.8 M NaC1, 30 mM MgC12, 20 mM CaC12, 8 mM HEPES (pH 7.0). An equal volume of sterile 60% polyethylene glycol (Fisher PEG 6000) was added, and the mixture was incubated 12-16 hours at 4~Cells were plated at 2.5• 105/100-mm dish 48 hours prior to transformation and refed with growth medium (Dulbecco's modified Eagle's medium [DMEM] with 10% horse serum) 20-24 hours prior to the addition of the PEG-precipitated phage. Medium was aspirated from the plates, and the cells were rinsed with phosphate-buffered saline; 1.1 ml of the phage suspension was added to the cells, together with l ml of prewarmed DMEM without serum. After incubation at 37~ for 15 minutes, an additional 2 ml of DMEM without serum was added. Incubation was continued at 37~ for 60 minutes, then l0 ml of growth medium was added. After 4 hours and again at 20 hours, the medium was replaced