Establishment Of The Minigene Method For Reporting The Alternative Splicing Of Bcl-x

Objective:Alternative splicing is a process by which the exons of the RNA produced by pre-mRNA are reconnected in multiple ways during RNA splicing. The resulting different mRNAs may be translated into different protein isoforms; thus, a single gene may code for multiple proteins.All human genes contain a diverse array of cis-acting elements within introns and exons that are required for correct and efficient precursor messenger RNA(pre-mRNA)splicing. The abnormal splicing may lead to some diseases. Minigene means that the relevant genomic DNA fragment is cloned within a plasmid between the promoter and the stop code, the recombinant plasmid can transcribe mRNA in eucaryotic cells and splice into different mRNA in different cell status. The main use of minigene is to detect mRNA alternation of alternative splicing in different cell status, it is a classical method for studing the mechanism of alternative splicing. Human apoptostic gene bcl-x alternative splicing abnormalities is closely related to a variety of tumor development. Here we constructed the human apopotosis-related Bcl-x minigene and its mutated minigene, and established the minigene method for reporting the alternative splicing of Bcl-x, to provide basis for studing the alternative splicing of Bcl-x gene.Methods:1.Amplification of the aim genomic DNA fragment by PCR: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,;secondly, the fragment (P3P4) including 3'sequence of intron 2 and part of exon 3 in bcl-x gene were amplified by PCR;2 Construction of bcl-x minigene by using directinally clone:pcDNA3.1(-) and P1P2 are cutted by double enzyme(XbaI, Xhol), and then retrieve the two fragments and ligated by T4 ligase,it is pcDNA3.1(-)-p1p2, and then transducted into DH5 a, we select the positive bacterial colony and purification of the plasmid, we idtntify the plasmid by double enzyme cut experiment and sequencing. We confirm that the sequence is all right by sequecing, the next we do is to cut the pcDNA3.1(-)-p1p2 and P3P4, and then retrieve the two fragments and ligated by T4 ligase,it is pcDNA3.1(-)-plp2-p3p4,it is pcDNA3.1(-)-bcl-x, we idtntify the plasmid by double enzyme cut experiment and sequencing;3. Construction of bcl-x cis-elements:B2G,CRCEl,CRCE2-related mutated minigene by inverse PCR:we design the primer for mutating the cis-elements and using the pcDNA3.1(-)-bcl-x as the template to conduct inverse PCR, and then we use the enzyme Dpnl to treat the PCR products, and make it self- cyclization, we use the product to transduct the competent cell DH5 a, select the positive bacterial colony and purification of the plasmid, we idtntify the plasmid by enzyme cut experiment and sequencing;4.Detection of the transcribe status of bcl-x minigene and its mutated minigene in HL60 cell by RT-PCR:Transfect the bcl-x minigene and its mutated minigene to HL60 cell respectively by Lipofectamine 2000, After 48h later, extracting the total RNA by TRNzol, and reverse-transcribed to cDNA, and using the specific primer to conduct PCR reaction. We detect the ratio of bcl-xL/bcl-xS.Results:(1) We amplified the correct genomic DNA fragment by detection with Agrose gel electrophoresis;(2) We did not find any fault mutated bases in the pcDNA3.1(-)-bcl-x minigene by double restricted enzyme cut experiment and sequencing, the bcl-x minigene can transcribe into mRNA in cell HL60 after transient tansfection.(3) We did not find any fault mutated bases in the mutated minigene: pcDNA3.1(-)-bcl-x-CRCE1(M),pcDNA3.1(-)-bcl-x-CRCE2(M) pcDNA3.1(-)-bcl-x-B2G(M), the bcl-x mutated minigene can transcribe successfully in cell HL60 after transient tansfection.(4) The results of RT-PCR indicate that:bcl-xL/bcl-xS is1.4 after transfection with pcDNA3.1(-)-bcl-x.We found that the cis-elements:CRCE1,CRCE2,B2G.had some influence on bcl-xS/bcl-xL mRNA by RT-PCR, the results of RT-PCR indicate that the bcl-xS has almost disspeared in HL60 after transfecting the mutated minigenes pcDNA3.1(-)-bcl-x-B2G(M),pcDNA3.1(-)-bcl-x-CRCE1(M); And the ratio of bcl-xL/bcl-xS is 1.6 in cell HL60 after transfecting the mutated minigene pcDNA3.1(-)-bcl-x-CRCE2(M).Conclusion:1.we amplified successfully the bcl-x genomic DNA fragment(P1P2,P3P4) including bcl-xS,bcl-xL splicing sites and some important cis-elements.2.We successfully construct the minigene by using directionally clone method and mutated minigene of human apoptotic-related gene bcl-x by using inverse PCR, and establish the minigene report method for studing the mechanism of bcl-x alternative splicing.3.the bcl-x minigene and the mutated minigene can transcribe correctly in HL60 cells, and the cis-elements:CRCE1,B2G mutated can make the bcl-xS almost disspeared; the mutated cis-element CRCE2 can increase the ratio of bcl-xL/bcl-xS in HL60 cell.