Requirement for the large T and small T proteins of SV40 in the maintenance of the transformed state.
Requirement for the large T and small T proteins of SV40 in the maintenance of the transformed state.
复制标题
维持转化状态需要 SV40 的大 T 和小 T 蛋白。
DOI：
10.1101/sqb.1980.044.01.037
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
Topp,WC
中科院分区：
文献类型：
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作者：
Frisque,RJ;Rifkin,DB;Topp,WC
材料和方法将776、d1884、d1890(来自康涅狄格大学健康中心法明顿分校T.Shenk)、d12008和tsA58(来自纽约州立大学Stony Brook的P.Tegtmeyer赠送)菌株从分离的空斑中培养，在CV-1细胞上进行增殖和滴定。所有病毒滴度均在1×10~(-1)~2×10~(-1)S pfu/ml之间，TsA58滴度在40.5~32.5时至少低4个对数。除非另有说明，否则细胞在含10%胎牛血清的Dulbecco改良Eagle‘s培养液(DMEM；GIBCO)中，在10%CO_2/空气的潮湿气氛中进行增殖。孵化器温度使用热电偶计(黄泉仪)仔细调节，并为整个实验保留了一批血清。胚胎培养是将近交系Fisher大鼠或LHC叙利亚仓鼠胚胎在妊娠15天时物理解聚，然后在37℃下胰酶消化约15分钟获得的。将培养物传代一次或两次，直到均匀，然后在液氮中冷冻。接种后1天，通过感染指数增长的培养物分离出每细胞约50pfu的病毒。第二天，被感染的培养物被胰酶消化并连续稀释。培养10d(37.5~3周)或3周(32.5~32.5个)，用钢制克隆圆柱体包裹分离出的菌落，在微孔(Linbro)中传代培养(1个细胞/孔)。用间接免疫荧光法检测单细胞转化株的病毒T抗原，并进行冰冻。所有实验都是在培养不到2周的新鲜复苏的、不含PPLO的品系上进行的。在装有模拟感染细胞的平板上，没有出现可以继代培养的菌落。
MATERIALS AND METHODSSV40 strains 776, d1884, d1890 (gifts from T. Shenk, University of Connecticut Health Center, Farmington), d12008, and tsA58 (a gift from P. Tegtmeyer, State University of New York, Stony Brook) were grown from isolated plaques, propagated, and titered on CV-1 cells. All virus stocks were in the range of 1 x los to 2 x l0 s pfu/ml, tsA58 titered at least four logs lower at 40.5~ than at 32.5~ Cells were propagated at 37.5~(unless otherwise stated) in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% fetal calf serum (FCS; Irvine) on plastic culture dishes (Falcon) in a humidified atmosphere of 10% CO2/air. Incubator temperatures were carefully regulated using a thermocouple gauge (Yellow Springs Instrument), and a single batch of serum was reserved for the entire experiment. Embryo cultures were obtained by physical disaggregation of either inbred Fisher rat or LHC Syrian hamster embryos, taken at 15 days of gestation, followed by trypsinization at 37~ for approximately 15 minutes. Cultures were passaged once or twice until homogeneous and then frozen in liquid nitrogen.Transformants were isolated by infection of exponentially growing cultures with approximately 50 pfu of virus per cell 1 day after seeding. The next day, the infected cultures were trypsinized and serially diluted. After incubation for 10 days (37.5~ or 3 weeks (32.5~ isolated colonies appearing at high dilution were ringed with steel cloning cylinders and subcultured (1 cell/well) in microwells (Linbro). Transformed lines thus derived from single cells were examined for viral T antigen by indirect immunofluorescence and frozen. All experiments were carried out on freshly revived, PPLO-free lines after less than 2 weeks in culture. No colonies that could be subcultured appeared on plates with mock-infected cells.