CELL-TRANSFORMING ABILITY OF A TEMPERATURE-SENSITIVE MUTANT OF POLYOMA VIRUS
CELL-TRANSFORMING ABILITY OF A TEMPERATURE-SENSITIVE MUTANT OF POLYOMA VIRUS
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DOI：
10.1073/pnas.53.3.486
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发表时间：
1965-01-01
影响因子：
11.1
通讯作者：
FRIED, M
中科院分区：
文献类型：
--
作者：
FRIED, M
材料与方法。-病毒株：多瘤病毒（PY）的亲本大空斑株已在其他地方描述过。4从一株PY砧木中分离到一株温度敏感突变体TS-a。细胞培养：BHK/C13仓鼠细胞是从苏格兰格拉斯哥病毒学研究所的Michael Stoker博士那里获得的，并一直在含有10%小牛血清和10%色氨酸磷酸肉汤的强化Eagle培养基中培养。转化试验：转化试验基本上是由Montagnier和Macpherson7'8描述的，通过在琼脂中形成菌落的增强能力来区分多瘤病毒转化的细胞与正常细胞。将Hamster细胞（BHK/C13）以108个细胞/ml的浓度在38.50℃的强化Eagle培养基中悬浮感染1小时，用强化Eagle培养基洗涤3次以去除未吸收的病毒，然后在含有10%小牛血清和10%磷酸色氨酸肉液的0.33%琼脂中以104个细胞/ml的浓度悬浮在强化Eagle培养基中。将1.5 ml的0.33%琼脂细胞悬浮液镀在预先设置的5 ml 0.5%琼脂的基础层上，置于60 mm的塑料培养皿中。琼脂硬化后，培养皿在31.50和/或38.50℃下在含有7%二氧化碳的良好湿润的培养箱中孵育。需要指出的是，在这些条件下，如Montagnier和Macpherson最初建议的那样，在38.50 C下播种超过2-3 × 104个细胞/皿，在38.50 C下孵育时，产生了许多正常细胞菌落。孵育条件：将实验皿分成四组，每组在进行转化前以以下方式之一孵育：(1)高温孵育-在38.50 C下孵育16-18天；(2)“上移”孵育——先在31.50℃下孵育4 d，然后在38.50℃下孵育16-18 d；(3)“下移”孵育——先在38.50℃下孵育2 d，再在31.50℃下孵育32-36 d；(4)低温孵育-在31.50℃下孵育32-36天。
Materials and Methods.-Virus strains: The parental large plaque strain of polyoma virus (PY) has been described elsewhere. 4 The temperature-sensitive mutant (TS-a) was isolated from a stock of PY after nitrous acid treatment. 3Cell culture: BHK/C13 hamster cells5 were obtained from Dr. Michael Stoker of the Institute of Virology in Glasgow, Scotland, and were always cultivated in reinforced6 Eagle's medium con-taining 10% calf serum and 10% tryptone phosphate broth. Transformation assay: The transformationassay was essentially that described by Montagnier and Macpherson7'8 which distinguishes the cells transformed by polyoma virus from normal cells by their enhanced ability to form colonies in agar. Hamster cells (BHK/C13) were infected in suspension at a concentration of 108 cells/ml in reinforced Eagle's medium for1 hr at 38.50 C. The infected cells were washed three times with reinforced Eagle's medium to remove the unabsorbed virus and then suspended in 0.33% agar containing 10% calf serum and 10% tryptone phosphate broth in reinforced Eagle's medium at a concentration of 104 cells/ml. One and one-half ml of the 0.33% agar cell suspension was plated onto a preset base layer of 5 ml of 0.5% agar in 60-mm plastic Petri dishes. After the agar had hardened, the dishes were incubated at 31.50 and/or 38.50 C in well-humidified incubators containing 7% CO2. It should be pointed out that under these conditions the seeding of more than 2-3 X 104 cells/dish, as originally suggested by Montagnier and Macpherson, 7 8 resulted in the production of many normal cell colonies when incubated at 38.50 C. Incubation conditions: The assay dishes were divided into four equal groups which were each incubated in one of the following fashions before scoring for transformation:(1) high-temperature incubation-incubation 16-18 days at 38.50 C;(2)" shift up" incubation-incubation first for4 days at 31.50 C and then 16-18 days at 38.50 C;(3)" shift down" incubation-incubation first for 2 days at 38.50 C and then 32-36 days at 31.50 C;(4) low-temperature incubation-incubation 32-36 days at 31.50 C.