生物
互补DNA
逆转录病毒
克隆(编程)
遗传学
分子生物学
基因
人类白细胞抗原
编码区
发起人
病毒学
基因表达
抗原
计算机科学
程序设计语言
作者
Yoshiki Akatsuka,Tracie A. Goldberg,Eisei Kondo,Emily Martin,Yuichi Obata,Yasuo Morishima,Takashi Takahashi,John A. Hansen
标识
DOI:10.1034/j.1399-0039.2002.590607.x
摘要
Abstract: Analysis of HLA restriction specificity is one of the important steps in characterizing T cell clones. This usually requires either a panel of HLA‐typed cells or HLA cDNA transfectants. Although preparation of HLA cDNA transfectants is laborious, utilization of transfectants is advantageous when a suitable panel is not available due to linkage disequilibrium or rarity of the HLA allele of interest. In this report, we describe an efficient and rapid HLA cloning and expression system. Three sets of PCR primers specific for HLA‐A, B and C loci were designed by extensively sequencing 5′‐ and 3′‐ untranslated regions of HLA class I genes. The PCR‐amplified products were introduced into modified Phoenix retrovirus vectors containing a puromycin resistant gene under the control of a LTR promotor. Gibbon ape leukemia virus (GALV)‐pseudotyped retrovirus was produced and infected into B‐lymphoid cell lines. Following expansion in selection media, more than 80% of cells expressed transduced HLA at a comparable level to that normally expressed. These results indicate that locus‐specific PCR cloning and utilization of GALV‐pseudotyped retroviral vector can be an effective and relatively efficient tool for constructing a panel of different HLA transfectants.
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