青鳉p53基因克隆、结构分析及同源重组载体构建

CLONING, STRUCTURAL ANALYSIS AND CONSTRUCTION OF HOMOLOGOUS RECOMBINATION VECTOR OF P53 GENE IN MEDAKA FISH (ORYZIAS LATIPES)

应用“Long PCR”技术 ,用 6对 p5 3引物从青胚胎干细胞基因组DNA中扩增出 6个相互重叠的片段 ,其中最大的片段长达 4 5kb ,这 6个PCR片段覆盖了整个 p5 3基因。序列分析表明青p5 3基因长约 8 7kb ,由 11个外显子和 10个内含子组成。结构比较表明 ,青 p5 3基因在大小上与人和小鼠 p5 3基因存在较大差异。青p5 3基因的内含子 1仅为 0 85kb ,而人和小鼠p5 3基因的内含子 1则分别长达 10kb和 6kb ;青 p5 3基因的外显子 3(86bp)明显大于人和小鼠 p5 3基因的外显子 3(2 2bp) ;外显子 4 (170bp)比人 (2 80bp)和小鼠 (2 6 0bp)的外显子 4小 ;内含子 10 (3 5kb)则比人和小鼠内含子 10 (0 7kb和 0 9kb)大得多。用SVTK neo基因作正选择标记基因 ,用SVTK tk基因作负选择标记基因 ,用青 p5 3基因组片段作同源序列 ,构建了鱼类 p5 3基因同源重组载体。将此载体转染青胚胎干细胞 ,并经G4 18和Ganc药物选择后证明上述正、负选择标记基因在干细胞中能够有效表达 ,并提供对G4 18的抗性和对Ganc的敏感性。

b The tumor suppressor p53 is critical for guarding the genome from the incorporation of damaged DNA. In mammals p53 functions in the regulation of the cell cycle, cell death and differentiation. Mutation of the p53 gene can lead to cell transformation and neoplasia. Similarly, cancer is also a frequent disease in fish. In analogy to the mammalian situation fish p53 was expected to play a similar crucial role in tumorigenesis. However, by inspecting p53 cDNA sequences from tumors in different fish species, surprisingly not a single mutation was found. We have therefore considered the possibility that p53, unlike many other molecules which appear to play the same role in tumor development in higher and lower vertebrates, might have a different function in fish. The p53 gene was cloned from the medaka fish ( Oryzias latipes) using the “long PCR' technique. Genomic DNA was isolated from the medaka embryonic stem cell like cell line MES1 according to the standard procedure. Six pairs of overlapping primers (N8 C2, N1 C8, N15 C9, N14 C11, N21 C10 and N28 C1) were used to amplify the medaka p53 gene were as follows: N1,5′ CAT GGA TCC TGT ACC CGA CCT G 3′;N8, 5′ ATC GAA TTC TGC AGT CGC CGA TTC AAA ATA TT 3′;N14, ATC GAA TTC AGT GTT ACA GTT CCT TAT GAG C 3′; N15, 5′ ATC GGA TCC CAT TCT CAG GGC CAC TGC GGT CTA 3′; N21, 5′ ATC GGA TCC TTC TTC TTT TCC CAG ATC AAC CTT 3′; N28, 5′ ATC GGA TCC GGA AAT TTC ATC AGC TCA GTA AC 3′; C1,5′ ATC TCT AGA CTT TAT TTA ACA AGG AAT TTG GTA C 3′; C2, 5′ CAG GTC GGG TAC AGG ATC CAT G 3′; C8, 5′ ATC TCT AGA CCA CGT GCT CCG TCT TCT TGT AGA 3′; C9, 5′ ATC TCT AGA GAG CAG GAT GGT GGT CAT CTC AGA 3′; C10, 5′ GGG TCT AGA TTG TTC CAT GCA TGT GTT CTT TAA T 3′; C11, 5′ CTA TCT AGA GCA CAG ATT CTG ACC TCG AA 3′. PCR products were cloned into the pBluescript KSⅡ+ vector and sequenced with the automated Alfexpress DNA analysis system. Sequence analysis revealed that the medaka p53 gene is 8 745 bp in length and consists of 11 exons. Comparison of the genomic organization of the p53 gene uncovered several differences between medaka and mammalian p53 genes: (1) Intron 1 (0 85 kb) of the medaka p53 gene is much smaller than that from the mouse (6 kb) and human (10 kb) p53 gene; (2) exon 3 (86 bp) is significantly larger than that of mammals (22 bp); (3) intron 10 (3 5 kb) in medaka p53 is 4 to 5 times larger than that in mice and humans. These data suggest that the medaka p53 gene differs from that in mice and humans in gene structure, especially in intron size. As the introns of the p53 gene in mammals contain important regulation elements and define some species specificity, these structural differences in the sizes of introns might point to a functional difference in transcriptional control. For studying the function of p53 in fish, homologous recombination vectors were constructed on the basis of the neo and tk cassettes and a genomic fragment of p53 in multiple steps. First, two fragments (2 kb and 4 8 kb) of medaka p53 gene were amplified by Long PCR, and ligated into basic plasmid pBluescript KSⅡ+, resulting in plasmid pM53N32C10 (9 8 kb). Secondly, the 1 2 kb neo cassette gene was inserted at the unique Bam HⅠ site between the 2 kb and the 4 8 kb fragments in pM53N32C10 as positive selection marker. The insertion disrupts the open reading frame of the p53 gene. The plasmid in which the orientation of neo was the same as that of the p53 gene was designated as pM53+ neo (11 kb). Finally, the 2 6 kb tk cassette was inserted at the Xba Ⅰ site in pM53+ neo as a negative selection marker, generating homologous recombination vector pM53+N K(13 6 kb). For transfection experiments, the plasmids pM53+N K were linearized with Not Ⅰ and transfected by electroporation. MES1 cells were plated in 10 cm dishes at a density of 80% confluence the day before electroporation. Cells of 350 μl at 1 8×10 7 cells/ml were electroporated in the presence of 48 μɡ of plasmid DNA using a single pulse delivered