Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (2): 289-293.doi: 10.3969/j.issn.1673-8225.2010.02.023

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Construction and identification of Bcl-x minigene and its mutated minigene

Huang Bo1, Wang Xiao-zhong1, Wang Cai-wen1, Li Jing2, Zhang Hai-bin1, Xiong Huo-mei1, Xiao Yun1   

  1. 1 Department of Laboratory Medicine, Second Affiliated Hospital of Nanchang University, Nanchang   330006, Jiangxi Province, China; 2 Department of Laboratory Medicine, First Affiliated Hospital of Nanchang University, Nanchang   330006, Jiangxi Province, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Wang Xiao-zhong, Associate professor, Department of Laboratory Medicine, Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China wangxzlj@126.com
  • About author:Huang Bo★, Studying for master’s degree, Department of Laboratory Medicine, Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China jxmuhb666@sohu.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30700338*

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Abstract:

BACKGROUND: It is not elucidated about its mechanism of the rising ratio of Bcl-xL/Bcl-xS linked closely with the development of tumors. The Bcl-x minigene is an important tool to study its mechanism of alternative splicing.
OBJECTIVE: To construct the human apoptosis-related Bcl-x minigene and its mutated minigene, and to provide basis for studying the alternative splicing of Bcl-x gene.
METHODS: At First, the fragment (P1P2) including exon 2 and part of 5’ sequence of intron 2 in bcl-x gene were amplified by PCR from human leukemia cell K562 genomic DNA, then directionally inserted into the eukaryotic expression vector pcDNA3.1(-), called pcDNA3.1(-)-p1p2; secondly, the fragment (P3P4) including 3’ sequence of intron 2 and part of exon 3 in bcl-x gene were amplified, then directionally inserted into the downstream of fragment p1p2, called pcDNA3.1(-)-p1p2-p3p4, it is pcDNA3.1(-)-bcl-x minigene. By sequencing and identifying, pcDNA3.1(-)-bcl-x minigene was introduced into HL-60 by transient transfection, so as to identify its expression by RT-PCR. In addition, pcDNA3.1(-)-bcl-x minigene was utilized as a template to construct its mutated minigene called pcDNA3.1(-)-bcl-x-CRCE1 (M) by inverse PCR.
RESULTS AND CONCLUSION: Taking human leukemia cell K562 genome DNA as the template, P1 and P2 as primers, approximately 686-bp target fragment were obtained by PCR amplification; taking P3 and P4 as primers, approximately 178-bp target fragment could be obtained. Through XbaⅠ and Eco RⅠdouble enzyme digestion, Xba Ⅰ and Xho Ⅰ double enzyme digestion, the anticipated fragment could be obtained from minigene pcDNA3.1(-)-bcl-x, also from mutated minigene pcDNA3.1(-)-bcl-x-CRCE1(M) through Eco RI single enzyme digestion, the sequence result was correct. Results showed that we successfully construct the minigene and mutated minigene of human apoptotic-related gene bcl-x. 

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