高效与特异的基因组定点修饰是基因工程动物研究的前沿与难点.链霉菌噬菌体ΦC31整合酶能介导含attB位点的外源基因定点整合于多种真核生物基因组的假attP位点,可维持外源基因的正常结构及高效表达.本文探讨ΦC31整合酶介导外源基因在...高效与特异的基因组定点修饰是基因工程动物研究的前沿与难点.链霉菌噬菌体ΦC31整合酶能介导含attB位点的外源基因定点整合于多种真核生物基因组的假attP位点,可维持外源基因的正常结构及高效表达.本文探讨ΦC31整合酶介导外源基因在猪基因组内定点整合的分子基础.构建含attB位点的报告载体pEGFP-N1-attB,与ΦC31整合酶表达载体pCMV-INT共转染猪肾PK15细胞,G418筛选获得单克隆细胞系.实时荧光定量PCR筛选出单拷贝整合的转基因细胞系.TAIL-PCR鉴定出1个猪基因组假attP位点,位于猪1号染色体,watson链,坐标114220087-114220126,命名为pig-attP-1.测序结果显示,pEGFP-N1-attB在attB位点处断开插入到pig-attP-1.用荧光计测定细胞培养基上清EGFP含量发现,该转基因细胞系EGFP的表达水平是本底的50倍(13500 AU vs.280 AU),表明pig-attP-1是利于外源基因高效表达的"友好位点".该研究不仅为实现外源基因在猪基因组内的定点整合提供了新策略,也为创制基因工程猪、建立动物生物反应器等研究注入了新思路.展开更多
The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. To better understand the activity of φC31 integrase in the bovine genome...The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. To better understand the activity of φC31 integrase in the bovine genome, DNA sequences of 44 integration events were analyzed, and 32 pseudo attP sites were identified. The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site. Genomic DNA flanking these sites typically contained repetitive sequence elements, such as short and long interspersed repetitive elements. These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration. In addition, BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 （DLC1） gene. Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome. This study represents the first time that the sequence features of pseudo attP sites specific integrase system has great potential for applied modifications in the bovine genome were analyzed. We conclude that this site- of the bovine genome.展开更多
Intron splicing in eukaryotic organisms requires the interactions of five snRNAs and numerous different proteins in the spliceosome. Although the molecular mechanism behind splicing has been well studied, relatively l...Intron splicing in eukaryotic organisms requires the interactions of five snRNAs and numerous different proteins in the spliceosome. Although the molecular mechanism behind splicing has been well studied, relatively little is known about regulation of expression for these splicing factor proteins. One of these proteins is the evolutionarily-conserved Drosophila RNP-4F splicing assembly factor. This protein is transcribed from a single gene into two developmentally regulated mRNAs that differ in their 5’-UTR structure. In the longer isoform, known to be abundant in the developing fly central nervous system, a conserved retained intron which folds into a stem-loop has been implicated in expression control of the mRNA. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the ΦC31 system. The results confirm our previous observation that presence of the regulatory stem-loop enhances RNP-4F protein expression. However, in that study, the enhancement factor protein was not identified. We show here that overexpression of the RNP-4F transgene compared to the control results in additional translation, as indicated by the GFP reporter in the fluorescent images. These results are interpreted to show that RNP-4F protein acts back on its own mRNA 5’-UTR regulatory region via a feedback pathway to enhance protein synthesis in the developing fly central nervous system. A model is proposed to explain the molecular mechanism behind rnp-4f gene expression control.展开更多
To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase （SSR） systems （Cre-loxP, FIp-FRT, and φC31） have been used for tissue-specific gene knockout. With the combina...To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase （SSR） systems （Cre-loxP, FIp-FRT, and φC31） have been used for tissue-specific gene knockout. With the combination of an SSR system and inducible gene expression systems （tetracycline and tamoxifen）, stage-specific knockout and transgenic expression can be achieved. The application of this ＂SSR＋inducible＂ conditional tool to genomic manipulation can be extended in various ways. Alternatives to conditional gene targeting, such as conditional gene trapping, multipurpose conditional alleles, and conditional gene silencing, have been developed. SSR systems can also be used to construct precise disease models with point mutations and chromosomal abnormalities. With these exciting achievements, we are moving towards a new era in which the whole genome can be manipulated as we wish.展开更多
文摘高效与特异的基因组定点修饰是基因工程动物研究的前沿与难点.链霉菌噬菌体ΦC31整合酶能介导含attB位点的外源基因定点整合于多种真核生物基因组的假attP位点,可维持外源基因的正常结构及高效表达.本文探讨ΦC31整合酶介导外源基因在猪基因组内定点整合的分子基础.构建含attB位点的报告载体pEGFP-N1-attB,与ΦC31整合酶表达载体pCMV-INT共转染猪肾PK15细胞,G418筛选获得单克隆细胞系.实时荧光定量PCR筛选出单拷贝整合的转基因细胞系.TAIL-PCR鉴定出1个猪基因组假attP位点,位于猪1号染色体,watson链,坐标114220087-114220126,命名为pig-attP-1.测序结果显示,pEGFP-N1-attB在attB位点处断开插入到pig-attP-1.用荧光计测定细胞培养基上清EGFP含量发现,该转基因细胞系EGFP的表达水平是本底的50倍(13500 AU vs.280 AU),表明pig-attP-1是利于外源基因高效表达的"友好位点".该研究不仅为实现外源基因在猪基因组内的定点整合提供了新策略,也为创制基因工程猪、建立动物生物反应器等研究注入了新思路.
基金supported by the grants from the National Science and Technology Major Project of China(Nos. 2009ZX08010-018B and 2011ZX08007-004)State & Shanghai Leading Academic Discipline(B204)
文摘The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. To better understand the activity of φC31 integrase in the bovine genome, DNA sequences of 44 integration events were analyzed, and 32 pseudo attP sites were identified. The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site. Genomic DNA flanking these sites typically contained repetitive sequence elements, such as short and long interspersed repetitive elements. These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration. In addition, BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 （DLC1） gene. Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome. This study represents the first time that the sequence features of pseudo attP sites specific integrase system has great potential for applied modifications in the bovine genome were analyzed. We conclude that this site- of the bovine genome.
文摘Intron splicing in eukaryotic organisms requires the interactions of five snRNAs and numerous different proteins in the spliceosome. Although the molecular mechanism behind splicing has been well studied, relatively little is known about regulation of expression for these splicing factor proteins. One of these proteins is the evolutionarily-conserved Drosophila RNP-4F splicing assembly factor. This protein is transcribed from a single gene into two developmentally regulated mRNAs that differ in their 5’-UTR structure. In the longer isoform, known to be abundant in the developing fly central nervous system, a conserved retained intron which folds into a stem-loop has been implicated in expression control of the mRNA. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the ΦC31 system. The results confirm our previous observation that presence of the regulatory stem-loop enhances RNP-4F protein expression. However, in that study, the enhancement factor protein was not identified. We show here that overexpression of the RNP-4F transgene compared to the control results in additional translation, as indicated by the GFP reporter in the fluorescent images. These results are interpreted to show that RNP-4F protein acts back on its own mRNA 5’-UTR regulatory region via a feedback pathway to enhance protein synthesis in the developing fly central nervous system. A model is proposed to explain the molecular mechanism behind rnp-4f gene expression control.
基金Project supported by the National Natural Science Foundation of China(Nos.30871436,30973297,and 31171194)the National Basic Research Program(973)of China(No.2010CB945002)+1 种基金the Ministry of Education of China(No.200804220011)Shandong Provincial Science and Technology Key Program(No.2009GG10003039),China
文摘To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase （SSR） systems （Cre-loxP, FIp-FRT, and φC31） have been used for tissue-specific gene knockout. With the combination of an SSR system and inducible gene expression systems （tetracycline and tamoxifen）, stage-specific knockout and transgenic expression can be achieved. The application of this ＂SSR＋inducible＂ conditional tool to genomic manipulation can be extended in various ways. Alternatives to conditional gene targeting, such as conditional gene trapping, multipurpose conditional alleles, and conditional gene silencing, have been developed. SSR systems can also be used to construct precise disease models with point mutations and chromosomal abnormalities. With these exciting achievements, we are moving towards a new era in which the whole genome can be manipulated as we wish.
