High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.
High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.
复制标题
通过原生质体融合将克隆的单纯疱疹病毒 1 型序列高频转移至哺乳动物细胞。
DOI：
10.1128/mcb.1.8.743-752.1981
复制
发表时间：
1981
影响因子：
5.3
通讯作者：
Glorioso,JC
中科院分区：
文献类型：
--
作者：
Sandri-Goldin,RM;Goldin,AL;Levine,M;Glorioso,JC
Schaffner 的原生质体融合技术（W. Schaffner, Proc. Natl. Acad. Sci. U.S.A.77:2163–2167, 1980）已被用于将克隆的单纯疱疹病毒基因引入培养的哺乳动物细胞中。该技术涉及用溶菌酶消化细菌细胞壁以产生原生质体，然后通过聚乙二醇处理将原生质体与哺乳动物细胞融合。为了监测转移，在融合前用荧光染料异硫氰酸荧光素标记原生质体。融合后，超过 50% 的哺乳动物细胞发出荧光，表明细菌物质以高频率转移。通过原位杂交测量，质粒 pBR325 的转移发生频率为 1% 至 2%。通过间接免疫荧光检测，由 1 型单纯疱疹病毒（KOS 株）EcoRI 片段 F 组成的嵌合质粒在 pBR325 中的融合转移导致约 5% 的哺乳动物细胞中某些病毒基因组序列的表达。 300至500个Ltk-细胞中的一个与携带嵌合质粒pX1的原生质体融合后转化为TK+表型，该质粒由pBR322和编码单纯疱疹病毒1型胸苷激酶基因的BamHI片段组成。
The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A.77:2163–2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS)EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk−cell in 300 to 500 was transformed to the TK+phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and theBamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.