Recombinant gene expression in vivo within endothelial cells of the arterial wall

A technique for the transfer of endothelial cells and expression of recombinant genes in vivo could allow the introduction of proteins of therapeutic value in the management of cardiovascular diseases. Porcine endothelial cells expressing recombinant beta-galactosidase from a murine amphotropic retroviral vector were introduced with a catheter into denuded iliofemoral arteries of syngeneic animals. Arterial segments explanted 2 to 4 weeks later contained endothelial cells expressing beta-galactosidase, an indication that they were successfully implanted on the vessel wall.     

Correction of murine beta-thalassemia by gene transfer into the germ line

A murine beta-thalassemia was corrected by the transfer of cloned beta-globin genes into the mouse germ line. The cloned mouse beta maj-globin gene or the cloned human beta-globin gene was introduced into mice deficient in beta-globin synthesis because of a deletion of the beta maj-globin gene. Both introduced genes produced functional beta-globin chains, leading to a reduction in one case, and elimination in another case, of the anemia and associated abnormalities of the red blood cells.     

Transgenic monkeys produced by retroviral gene transfer into mature oocytes

Transgenic rhesus monkeys carrying the green fluorescent protein (GFP) gene were produced by injecting pseudotyped replication-defective retroviral vector into the perivitelline space of 224 mature rhesus oocytes, later fertilized by intracytoplasmic sperm injection. Of the three males born from 20 embryo transfers, one was transgenic when accessible tissues were assayed for transgene DNA and messenger RNA. All tissues that were studied from a fraternal set of twins, miscarried at 73 days, carried the transgene, as confirmed by Southern analyses, and the GFP transgene reporter was detected by both direct and indirect fluorescence imaging.     

节节麦抗白粉病基因直接转移及遗传表达

利用四呼推广高产小麦品种（系）和地方品种直接与抗、感白粉病节节麦杂交,通过幼胚培养技术成功获得２２个组合的杂种植株。将杂种Ｆ１及回交ＢＣ１Ｆ１与其普通小麦、节节麦亲本的抗白粉性比较分析,发现大部分组合中节节麦的抗性基因被普通小麦的抑制基因抑制,仅１份节节麦的抗性基因能在杂种Ｆ１中完全或部分表达;节节麦抗性基因的表达和抑制,与特定的普通小麦亲本有关。结果还表明,杂种Ｆ１中被抑制的节节麦抗性基因,可以通过用不含抑制基因的普通小麦回交,使其在回交后代中正常表达。     

Gene expression in mice after high efficiency retroviral-mediated gene transfer

A retroviral expression vector (N2) containing the selectable gene, neoR, has been used to determine the optimal conditions for infecting murine hematopoietic progenitor cells at high efficiency. After infected bone marrow cells were introduced into lethally irradiated mice, the presence, stability, and expression of the vector DNA sequences were analyzed either in individual spleen foci 10 days later or in the blood, bone marrow, and spleens of mice 4 months later. When bone marrow cells were cultured in medium containing virus with titers of more than 10(6) colony-forming units per milliliter in the presence of purified murine interleukin-3, more than 85 percent of the resulting foci contained vector DNA. This proviral vector DNA was intact. Efficient expression of the neoR gene was demonstrated in most of the DNA-positive foci examined. The spleens of reconstituted animals (over a long term) contained intact "vector DNA" and the blood and bone marrow expressed the neoR gene in some animals. Thus, a retroviral vector can be used to introduce intact exogenous DNA sequences into hematopoietic stem cells with high efficiency and with substantial expression.     

Retroviral vector-mediated gene transfer into human hematopoietic progenitor cells

The transfer of the human gene for hypoxanthine phosphoribosyltransferase (HPRT) into human bone marrow cells was accomplished by use of a retroviral vector. The cells were infected in vitro with a replication-incompetent murine retroviral vector that carried and expressed a mutant HPRT complementary DNA. The infected cells were superinfected with a helper virus and maintained in long-term culture. The production of progeny HPRT virus by the bone marrow cells was demonstrated with a colony formation assay on cultured HPRT-deficient, ouabain-resistant murine fibroblasts. Hematopoietic progenitor cells able to form colonies of granulocytes or macrophages (or both) in semisolid medium in the presence of colony stimulating factor were present in the nonadherent cell population. Colony forming units cloned in agar and subsequently cultured in liquid medium produced progeny HPRT virus, indicating infection of this class of hematopoietic progenitor cell.     

梨磷脂酰胆碱转移蛋白基因的克隆及其表达分析

据转录组测序结果,结合RACE(cDNA末端快速扩增技术)和Genome walking(染色体步移技术)获得了梨磷脂酰胆碱转移蛋白基因的完整读码框序列,其开放阅读框长为1 323 bp,编码的440个氨基酸序列与葡萄磷脂酰胆碱转移蛋白质的同源相似性达73%,其具有START domain(启动域)、ZnF_TAZ(TAZ锌指结构)、DCD(双氰胺)保守结构域,将梨磷脂酰胆碱转移蛋白基因命名为PePCTP1。PePCTP1基因与GFP融合,构建植物表达载体,洋葱表皮亚细胞定位观察结果显示,PePCTP1基因编码的蛋白质分布在细胞膜上。用激素生长素(IAA)、脱落酸(ABA)、水杨酸(SA)、赤霉素(GA)、细胞分裂素(6-BA)及NaCl诱导处理,发现与对照(CK)相比,SA和GA处理后PePCTP1基因表达量显著上升;NaCl和IAA处理后PePCTP1基因的表达量随时间逐渐上升;而ABA和6-BA处理不能增加目的基因的表达量。据此推测PePCTP1可能参与梨的逆境胁迫抗性反应。     

phlb基因对簇毛麦遗传物质向小麦直接遗传转移的影响

经人工授粉和幼胚培养成功地获得了普通小麦“中国春”CS及其phlb突变体CSphlb与簇毛麦(Haynaldia villosa)的属间杂种F     

Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues

The gene encoding human interleukin-2 (IL-2) has been cloned from human spleen cells, peripheral blood lymphocytes, and the Jurkat cell line. Nucleotide sequence analysis of the gene revealed that the encoded IL-2 protein has three cysteines located at amino acid residues 58, 105, and 125 of the mature protein. Site-specific mutagenesis procedures were used to modify the IL-2 gene by changing each of the cysteine codons individually to serine codons. Substitution of serine for cysteine residues at either position 58 or 105 of the IL-2 protein substantially reduced biological activity, indicating that the cysteines at these positions are necessary for maintenance of the biologically active conformation and may therefore be linked by a disulfide bridge. The modified IL-2 protein containing a substitution at position 125 retained full biological activity, suggesting that the cysteine at this position is not involved in a disulfide bond and that a free sulfhydryl group at that position is not necessary for receptor binding.     

实时荧光定量PCR筛选鸡基因表达最佳内参基因

为筛选在鸡基因表达时稳定表达的内参基因,本试验以6、14、22及30周龄的地方品种固始鸡与6周龄的商业品种罗斯308肉鸡为试验动物,以B2M、28S、GAPDH、β-actin及HSP70为候选内参基因,利用qRT-PCR对这5个候选内参基因的相对表达量进行测定,通过独立评价软件geNorm、NormFinder、SP...     

Chromosomal gene transfer in Spiroplasma citri

The study of resistance marker rearrangement in Spiroplasma citri mutants provides evidence of transfer of chromosomal information followed by recombination. This is the first report of such a transfer in vivo in the mollicutes--that is, in the smallest self-replicating organisms. The double-resistant phenotypes obtained are stable even without selection pressure. The mechanism of gene transfer is insensitive to deoxyribonuclease, requires contact, and possibly, areas of fusion of the cell membranes; it shares properties with the transfer by protoplast fusion in Gram-positive bacteria. The extensive degenerative evolution of mollicutes has retained, in S. citri, bacterial functions of chromosomal transfer and recombination.     

Regulation of chamber-specific gene expression in the developing heart by Irx4

The vertebrate heart consists of two types of chambers, the atria and the ventricles, which differ in their contractile and electrophysiological properties. Little is known of the molecular mechanisms by which these chambers are specified during embryogenesis. Here a chicken iroquois-related homeobox gene, Irx4, was identified that has a ventricle-restricted expression pattern at all stages of heart development. Irx4 protein was shown to regulate the chamber-specific expression of myosin isoforms by activating the expression of the ventricle myosin heavy chain-1 (VMHC1) and suppressing the expression of the atrial myosin heavy chain-1 (AMHC1) in the ventricles. Thus, Irx4 may play a critical role in establishing chamber-specific gene expression in the developing heart.     

多个环境因素对嗜水气单胞菌感染团头鲂致病力的影响

目的研究pH对拟态弧菌(Vibrio mimicus)感染相关表型及胞外产物特性的影响。方法测定不同pH (6.0、7.0、8.0、8.5和9.0)下拟态弧菌的增殖、泳动、自聚集和生物膜形成以及胞外产物(extracellular products,ECPs)的蛋白质量浓度、酶活性、溶血性、细胞毒性和毒力等特性。结果pH 6.0~9.0范围内,随着pH值升高,拟态弧菌的增殖先增强后减弱,pH 8.5时增殖最强;自聚集、生物膜形成和ECPs毒力先减弱后增强,pH 8.5时显著减弱(P<0.05);泳动以及ECPs蛋白质量浓度和细胞毒性减弱,pH 6.0时显著强于其他pH (P<0.05);ECPs酶活性和溶血性呈波动性,酶活性在pH 6.0时最强、pH 8.0时最弱,溶血性在pH 8.0时最强、pH 8.5时最弱。转录组分析显示：拟态弧菌pH 8.5较pH 7.0时,与ATP合成相关的糖酵解、TCA循环和氧化磷酸化等过程的基因表达上调,而与ADP合成相关的嘌呤合成基因、与蛋白质合成相关的核糖体亚基合成基因以及与蛋白分泌和膜运输相关的基因表达下调。结论pH对拟态弧菌感染相关表型及ECPs特性具有明显影响,且pH可能是通过调节拟态弧菌能量代谢与膜运输等过程影响其相关生物学特性。     

High frequency of horizontal gene transfer in the oceans

Oceanic bacteria perform many environmental functions, including biogeochemical cycling of many elements, metabolizing of greenhouse gases, functioning in oceanic food webs (microbial loop), and producing valuable natural products and viruses. We demonstrate that the widespread capability of marine bacteria to participate in horizontal gene transfer (HGT) in coastal and oceanic environments may be the result of gene transfer agents (GTAs), viral-like particles produced by α-Proteobacteria. We documented GTA-mediated gene transfer frequencies a thousand to a hundred million times higher than prior estimates of HGT in the oceans, with as high as 47% of the culturable natural microbial community confirmed as gene recipients. These findings suggest a plausible mechanism by which marine bacteria acquire novel traits, thus ensuring resilience in the face of environmental change.     

Correction of CD18-deficient lymphocytes by retrovirus-mediated gene transfer

Leukocyte adhesion deficiency (LAD) is an inherited disorder of leukocyte function caused by derangements in CD18 expression. The genetic and functional abnormalities in a lymphocyte cell line from a patient with LAD have been corrected by retrovirus-mediated transduction of a functional CD18 gene. Lymphocytes from patients with LAD were exposed to CD18-expressing retrovirus and enriched for cells that express CD11a and CD18 (LFA-1) on the cell surface. Molecular and functional analyses of these cells revealed (i) one copy of proviral sequence per cell, (ii) viral-directed CD18 RNA that exceeded normal endogenous levels, (iii) normal quantities of CD11a and CD18 protein on the cell surface, and (iv) reconstitution of LFA-1-dependent adhesive function.