PiggyBac转座元件的构建及其在团头鲂基因组中的转基因效率

鳞翅目昆虫粉纹夜蛾(Trichoplusia ni)的PiggyBac(PB)转座子已用于模式生物小鼠的转基因及插入诱变研究,目前,该转座子在养殖鱼类中的转基因效率如何还不清楚。构建了带PiggyBac转座子左臂、右臂、EF1α启动子和绿色荧光蛋白(eGFP)编码框的pPBs-EF1α-eGFP供体质粒。以50 ng/μL供体质粒和100 ng/μL体外转录的PB转座酶mRNA共同显微注射入团头鲂(Megalobrama amblycephala)1~2细胞期受精卵中,团头鲂eGFP的荧光表达率可达58.26%,PCR检测结果显示,该转座系统在团头鲂成鱼基因组中的整合效率为53.04%。表明PiggyBac转座子可高效介导基因在团头鲂基因组中的插入,为进一步开展团头鲂插入诱变研究奠定了基础。     

PiggyBac Transposon Mediated Efficient eGFP Expression in Porcine Somatic Cells and Cloned Embryos

PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac tr...     

Global transposon mutagenesis and a minimal Mycoplasma genome

Mycoplasma genitalium with 517 genes has the smallest gene complement of any independently replicating cell so far identified. Global transposon mutagenesis was used to identify nonessential genes in an effort to learn whether the naturally occurring gene complement is a true minimal genome under laboratory growth conditions. The positions of 2209 transposon insertions in the completely sequenced genomes of M. genitalium and its close relative M. pneumoniae were determined by sequencing across the junction of the transposon and the genomic DNA. These junctions defined 1354 distinct sites of insertion that were not lethal. The analysis suggests that 265 to 350 of the 480 protein-coding genes of M. genitalium are essential under laboratory growth conditions, including about 100 genes of unknown function.     

PiggyBac转座子介导的诱导细胞永生化转基因载体的构建及其整合效率的检测

【目的】构建在小鼠水平上实现多个目的基因的表达以及标记基因安全删除的转基因载体。【方法】以载体为模板,分别扩增SV40P neo、IRES、tk-PloyA,通过overlap PCR连接,并在两端添加方向相同的LoxP序列,构建转基因基本载体PB-NIT;然后通过overlap PCR扩增获得Tet-CMV-SV40 T-T2A-p 53-PolyA基因表达盒,将其插入PB-NIT中,构建载体PB-NIT-STP;最后将转录因子激活域 rtTA 通过同尾酶连接插入PB-NIT-STP,构建载体PB-rtTA-NIT-STP。【结果】经酶切和测序等鉴定,以上载体均正确;经转座活性鉴定,共转染转座子载体PB-rtTA-NIT-STP和转座酶载体比只转染转座子载体获得的阳性克隆数提高了20倍。【结论】得到了基于PiggyBac转座子的可用于正负向筛选和诱导目的基因表达的转基因载体。     

反刍月形单胞菌乙酸生成缺陷株的构建及相关酶活性分析(英文)

[目的]构建反刍月形单胞菌乙酸生成的缺陷株并分析其发酵特性。[方法]应用转座子标签法,通过转座子供体菌E.coliS17-1/pZJ25∷Tn5对受体菌反刍月形单胞菌进行转座子诱变,采用含卡那霉素和氟乙酸纳的选择性培养基筛选接合子。[结果]共筛选出稳定的对卡那霉素和氟乙酸具有抗性的转座工程菌7株。对反刍月形单胞菌的突变株进行16SrRNA鉴定和Tn5的PCR鉴定,及乙酸激酶(AK)和磷酸乙酰转移酶(PTA)酶比活力分析,确定突变株属于pta基因缺失型氟乙酸抗性菌株。[结论]为进一步研究反刍兽瘤胃微生物乙酸的细胞代谢网络和调控奠定基础。     

Rapid identification of nonessential genes of herpes simplex virus type 1 by Tn5 mutagenesis

The large genome of herpes simplex virus type of (HSV-1) encodes at least 80 polypeptides, the majority of which have no recognized function. A subgroup of these gene products appears to be nonessential for virus replication in cell culture, but contributes to the complex life cycle of the virus in the host. To identify such functions, a simple insertional mutagenesis method has been used for selective inactivation of individual HSV-1 genes. The bacterial transposon Tn5 was allowed to insert randomly into cloned restriction fragments representing the entire short unique (US) region of the HSV-1 genome. Of the 12 open reading frames that were mutagenized with Tn5, mutant derivatives of US2, US4, and US5 were recombined into the virus. These three genes proved to be nonessential for HSV-1 replication in Vero (African Green monkey kidney) cells and the US4 gene appeared to be involved in viral pathogenesis in the central nervous system of mice. This rapid mutagenesis procedure should prove useful in exploring the entire HSV-1 genome as well as the genomes of other complex animal viruses.     

Specific mutations induced by triplex-forming oligonucleotides in mice

Triplex-forming oligonucleotides (TFOs) recognize and bind to specific duplex DNA sequences and have been used extensively to modify gene function in cells. Although germ line mutations can be incorporated by means of embryonic stem cell technology, little progress has been made toward introducing mutations in somatic cells of living organisms. Here we demonstrate that TFOs can induce mutations at specific genomic sites in somatic cells of adult mice. Mutation detection was facilitated by the use of transgenic mice bearing chromosomal copies of the supF and cII reporter genes. Mice treated with a supF-targeted TFO displayed about fivefold greater mutation frequencies in the supF gene compared with mice treated with a scrambled sequence control oligomer. No mutagenesis was detected in the control gene (cII) with either oligonucleotide. These results demonstrate that site-specific, TFO-directed genome modification can be accomplished in intact animals.     

A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism

Human cytomegalovirus infects vascular tissues and has been associated with atherogenesis and coronary restenosis. Although established laboratory strains of human cytomegalovirus have lost the ability to grow on vascular endothelial cells, laboratory strains of murine cytomegalovirus retain this ability. With the use of a forward-genetic procedure involving random transposon mutagenesis and rapid phenotypic screening, we identified a murine cytomegalovirus gene governing endothelial cell tropism. This gene, M45, shares sequence homology to ribonucleotide reductase genes. Endothelial cells infected with M45-mutant viruses rapidly undergo apoptosis, suggesting that a viral strategy to evade destruction by cellular apoptosis is indispensable for viral growth in endothelial cells.     

PhoR/PhoP双因子调控系统在枯草芽孢杆菌NCD-2菌株解磷能力中的功能分析

枯草芽孢杆菌（Bacillus subtilis）NCD-2能有效的防治棉花黄萎病,同时还具有降解蛋黄卵磷脂的能力。将含有mini Tn10转座子的pHV1249质粒电击转入NCD-2菌株中,51℃高温诱导转座子插入突变,构建了NCD-2菌株的解磷突变子库,筛选到3株丧失解磷能力的突变子。运用染色体步移技术对突变株M216中转座子插入位点基因的侧翼序列进行克隆和序列分析,结果表明丧失解磷能力突变株中转座子插入基因与B.subtilis 168菌株中PhoR基因的相似率达到98%,Southern杂交验证突变株中转座子为单拷贝插入。将PhoR基因克隆到pHY300PLK质粒上构建重组质粒电击转化M216进行功能互补验证,互补菌株部分恢复了解磷能力,表明NCD 2菌株中PhoR基因与其降解卵磷脂能力具有相关性。     

利用双标记mTn5构建甲基杆菌MB200突变体库的研究

Methylobacterium sp. MB200是一种粉红色的、兼性甲基营养菌,它能以单碳化合物作为唯一碳源和能源,因此,可以作为氨基酸、工业酶等的生产菌株。以M. sp. MB200一碳代谢途径作为主要研究对象,利用mTn5-gusA-pgfp21转座子构建了甲醇利用菌的突变体库,在含有丁二酸的培养基上共获得了约12000株突变体,其中,甲醇营养缺陷的突变体119株,表现GUS活性的突变体1542株。然后,根据转座子已知序列,运用TAIL-PCR的方法克隆到了突变基因的部分序列,为进一步研究一碳代谢基因以及氨基酸和工业酶等的生产打下了基础。     

家蚕基因工程载体研究新进展

基因工程分为将外源DNA整合到宿主染色体的种系转移和外源DNA不整合到宿主染色体的瞬时表达,用于家蚕基因工程的载体主要有两类,一类是基于转座子的载体:Minos转座子载体、Mosl元件载体和PiggyBac转座子载体。另一类是基于病毒的载体:杆状病毒载体S、indbis病毒载体和假型反转录病毒载体。家蚕种系转移主要采用piggyBac转座子载体,而外源基因的表达则主要采用杆状病毒载体和家蚕培养细胞组成的NPV-Bm表达系统,Sindbis病毒载体在家蚕RNA干涉的研究中显示出了诱人的前景。     

BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity

Germline mutations of the breast cancer 1 (BRCA1) gene are a major cause of familial breast and ovarian cancer. The BRCA1 protein displays E3 ubiquitin ligase activity, and this enzymatic function is thought to be required for tumor suppression. To test this hypothesis, we generated mice that express an enzymatically defective Brca1. We found that this mutant Brca1 prevents tumor formation to the same degree as does wild-type Brca1 in three different genetically engineered mouse (GEM) models of cancer. In contrast, a mutation that ablates phosphoprotein recognition by the BRCA C terminus (BRCT) domains of BRCA1 elicits tumors in each of the three GEM models. Thus, BRCT phosphoprotein recognition, but not the E3 ligase activity, is required for BRCA1 tumor suppression.     

Modeling the initiation and progression of human acute leukemia in mice

Our understanding of leukemia development and progression has been hampered by the lack of in vivo models in which disease is initiated from primary human hematopoietic cells. We showed that upon transplantation into immunodeficient mice, primitive human hematopoietic cells expressing a mixed-lineage leukemia (MLL) fusion gene generated myeloid or lymphoid acute leukemias, with features that recapitulated human diseases. Analysis of serially transplanted mice revealed that the disease is sustained by leukemia-initiating cells (L-ICs) that have evolved over time from a primitive cell type with a germline immunoglobulin heavy chain (IgH) gene configuration to a cell type containing rearranged IgH genes. The L-ICs retained both myeloid and lymphoid lineage potential and remained responsive to microenvironmental cues. The properties of these cells provide a biological basis for several clinical hallmarks of MLL leukemias.     

Tissue-specific and ectopic expression of genes introduced into transgenic mice by retroviruses

Recombinant retroviruses containing the complete genomic human beta globin gene (under the control of its own promoter) and the bacterial neomycin phosphotransferase gene (under the control of the normal or enhancerless viral promoter) were used to derive transgenic mouse strains by infection of preimplantation embryos. Expression of the beta globin gene in hematopoietic tissues was observed in all transgenic strains. In addition, one strain showed ectopic expression of beta globin in the same tissues that also expressed high levels of RNA from the viral promoter. It is likely that expression from the long terminal repeat (LTR), in contrast to expression from the internal promoter, is dependent on the site of integration. Thus, retroviral vectors can be used for tissue-specific expression of foreign genes in transgenic mice, as well as for the identification of loci that allow developmental activation of a provirus.     

Hematopoietic stem cell quiescence maintained by p21cip1/waf1

Relative quiescence is a defining characteristic of hematopoietic stem cells, while their progeny have dramatic proliferative ability and inexorably move toward terminal differentiation. The quiescence of stem cells has been conjectured to be of critical biologic importance in protecting the stem cell compartment, which we directly assessed using mice engineered to be deficient in the G1 checkpoint regulator, cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21). In the absence of p21, hematopoietic stem cell proliferation and absolute number were increased under normal homeostatic conditions. Exposing the animals to cell cycle-specific myelotoxic injury resulted in premature death due to hematopoietic cell depletion. Further, self-renewal of primitive cells was impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure. Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.     

Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1

The Arabidopsis gene DDM1 is required to maintain DNA methylation levels and is responsible for transposon and transgene silencing. However, rather than encoding a DNA methyltransferase, DDM1 has similarity to the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes, suggesting an indirect role in DNA methylation. Here we show that DDM1 is also required to maintain histone H3 methylation patterns. In wild-type heterochromatin, transposons and silent genes are associated with histone H3 methylated at lysine 9, whereas known genes are preferentially associated with methylated lysine 4. In ddm1 heterochromatin, DNA methylation is lost, and methylation of lysine 9 is largely replaced by methylation of lysine 4. Because DNA methylation has recently been shown to depend on histone H3 lysine 9 methylation, our results suggest that transposon methylation may be guided by histone H3 methylation in plant genomes. This would account for the epigenetic inheritance of hypomethylated DNA once histone H3 methylation patterns are altered.     

The evolution of resistance gene in plants

Resistance genes enable plants to fight against plant pathogens. Plant resistance genes (R gene) are organized complexly in genome. Some resistance gene sequence data enable an insight into R gene structure and gene evolution. Some sites like Leucine-Rich Repeat (LRR) are of specific interest since homologous recombination can happen. Crossing over, transposon insertion and excision and mutation can produce new specificity. Three models explaining R gene evolution were discussed. More information needed for dissection of R gene evolution though some step can be inferred from genetic and sequence analysis.     

The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide

The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by Mycobacterium tuberculosis (Mtb). However, the protection is imperfect and infection persists. To identify genes that Mtb requires to resist RNI, we screened 10,100 Mtb transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. An Mtb mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type Mtb to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.