异种移植供体猪α-1,3GT基因打靶引导序列获得及其染色体定位

OBTAINING OF HOMOLOGOUS RECOMBINATION DIRECTING SEQUENCE AND CHROMOSE MAPPING OF PIG α-1,3GT GENE TARGETING IN XENOGNEIC DONOR ORGAN

目的 :为阻止异种抗原靶分子的生成 ,抑制或减轻HAR发生 ,试图对异种抗原的合成酶α - 1,3GT基因进行打靶灭活 ,以产生α - 1,3GT表型缺乏且具有抗HAR反应能力的异种供体 (猪 )。方法 :以α - 1,3GT基因的cDNA片段为探针 ,采用噬菌斑原位杂交结合PCR技术筛选猪gDNA文库 ,经酶切、Southern杂交、测序鉴定所得片段 ,并采用荧光原位杂交定位。结果 :获 7个片段 ,均在 8kb以上 ,且包含第三内含子 ,其中 3个片段测序证实包括第三、第四外显子 ;荧光原位杂交证实该基因位于猪染色体 1q2 .10～q2 .11。结论 :研究表明所得片段可作为该基因打靶引导序列 ,染色体定位明确了基因打靶位点 ,PCR技术可以用于快速筛选

Objective:To inhibit antigen production and prevent hyperacute rejection (HAR), we focus on the pig α-1,3 Galactosyltrasferase (α-1, 3GT) gene targeting for inhibiting HAR,because of the α-1,3GT is the synthetical enzyme of porcine xenogeneic antigen. Methods:We screened the porcine gDNA library by using α-1,3 GT cDNA fragment as a probe and Polymerase chain reaction(PCR), and adopt enzymic digestion, southern blot, sequencing to verify these postive inserts,Meanwhile Fluorescence in situ hybridization(FISH) was performed. Results:Our results showed that every insert length of 7 positive clone is above 8kb, including the third intron, Moreover 3 clone among them contain the third and fourth exons according to the sequencing results, and FISH mapped the inserts of the 3 clones to pig chromose 1q2.10～q2.11.Conclusion:The current study suggested that these gDNA fragments we obtained can be used as homologous recombination direction sequence (HRDS) in α-1,3 GT gene targeting ,FISH location of α-1,3 GT provided us gene targeting orientation; and PCR may be use as an effective method in screening DNA library.