EFFECT OF VIRUSES ON EARLY MAMMALIAN DEVELOPMENT .1. ACTION OF MENGO ENCEPHALITIS VIRUS ON MOUSE OVA CULTIVATED IN VITRO
EFFECT OF VIRUSES ON EARLY MAMMALIAN DEVELOPMENT .1. ACTION OF MENGO ENCEPHALITIS VIRUS ON MOUSE OVA CULTIVATED IN VITRO
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DOI：
10.1073/pnas.50.3.576
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发表时间：
1963-01-01
影响因子：
11.1
通讯作者：
GWATKIN, RBL
中科院分区：
文献类型：
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作者：
GWATKIN, RBL
材料和方法：随机饲养6-8周龄的瑞士小鼠，通过腹腔注射5IU的孕马血清促性腺激素(Gestyl，Organon)进行超数排卵，43小时后再注射5IU。人绒毛膜促性腺激素(孕酮，有机磷)。在第二次注射时，每只雌性都放置了一只成熟的雄性动物。这种治疗会导致排卵和交配大约12小时后。有阴道塞子的雌性(通常是70%-90%的动物)在34-36小时后被杀，即第一次卵裂预期时间后10-12小时。用带有钝的30号针头的注射器将2-细胞卵子从输卵管中冲洗出来。用来冲洗卵子和培养它们的介质是由RL Brinster博士开发的，并将在其他地方发表。5用改良的Krebs-Ringer平衡盐溶液，添加乳酸钠和结晶牛血浆蛋白。为了确定透明带是否是病毒入侵的屏障，用灰色链霉菌蛋白酶(Pronase，CalBiochem Co.)去除透明带。6将卵母细胞置于含1.0%聚乙烯吡咯烷酮(PVP)的0.25%“Pronase”磷酸盐缓冲液中，室温暴露5~10min。添加PVP是为了保护凹陷的卵裂球，并防止它们附着在玻璃上。“Pronase”和其他酶在老鼠卵的透明带上的作用的更多细节在其他地方描述。7将裸卵或卵带完整的卵子放入含或不含病毒或抗血清的乳酸-白蛋白培养液中。将这些液滴浸入60毫米的培养皿中的矿物油中，以便在防止蒸发的同时进行气体交换。培养皿在370℃，空气中二氧化碳含量为5%的气氛中孵化。用Brownstein和Graham分离的Mengo脑炎病毒37A(热稳定性突变体)，用作者描述的空斑技术对病毒进行了鉴定。
Materials and Methods.-Randomly bred 6-to 8-week-old Swiss mice were superovulated by the intraperitoneal injection of 5 IU of pregnant mare serum gonadotrophin (Gestyl, Organon), followed 43 hr later by 5 LU. of human chorionic gonadotrophin (Pregnyl, Organon). A mature male was placed with each female at the time of the second injection. This treatment results in ovulation and mating about 12 hrafterwards. Females with vaginal plugs (usually 70-90% of the animals) were killed 34-36 hr later, that is, 10-12 hr after the expected time of the first cleavage division. The 2-cell eggs were flushed from the Fallopian tubes using a syringe with a blunted no. 30needle. The medium used for flushing out the eggs and also for culturing them was de-veloped by Dr. RL Brinster and isto be published elsewhere. 5 This medium consists of modified Krebs-Ringer balanced salt solution, supplemented with sodium lactate and crystalline bovine plasma albumin.To determine whether the zona pellucida was a barrier to viral entry it was removed with Streptomyces griseus protease (" Pronase," Calbiochem Co.). 6 Ova were exposed 5-10 min at room temperature to 0.25%" Pronase" in phosphate buffered saline, containing 1.0% polyvinyl-pyrrolidone (PVP). The PVP was added to protect thenaked blastomeres and to preventtheir attachment to the glass. Further details of the action of" Pronase," and other enzymes, on the zona pellucida of the mouse egg are described elsewhere. 7 The eggs, either naked or with their zonae intact, were placed in drops of the lactate-albumin medium, with or without virus or antiserum. The drops were submerged in mineral oil in a 60 mm Petri dish to permit gas-exchange while preventing evaporation. The culture dishes were incubated at 370C in an atmosphere of 5% CO2 in air. The 37A (heat-stable mutant) of Mengo encephalitis virus, isolated by Brownstein and Graham,'was used. The virus was assayed by the plaque technique decribed by these authors.