Patterns of Integration and Expression of Retroviral, Non-Replicative Vectors in Avian Embryos: Embryo Developmental Stage and Virus Subgroup Envelope Modulate Tissue-Tropism

We previously demonstrated that Avian Leukemia Viruses (ALV) carrying the v-myc gene specifically induce two types of tumors, cardiomyocytic tumors when the virus is injected before embryonic day 3 (E3), skin tumors when the virus is injected at E3 or E5.

Aiming to elucidate the mechanisms which determine this time-dependent change in target, we infected chick and quail embryos at E3 and E5 with replication-deficient, lacZ gene-carrying, ALV-based viruses produced by a packaging cell line. Three constructs driven by 3 different Long Terminal Repeats (LTRs) were tested and yielded similar results. When the constructs were inoculated at E3 and the lacZ gene product revealed 5 days later, around 70% of the embryos carried lacZ+ clones in the heart, around 50% had positive clones in the skin anywhere on the body, while a few embryos displayed clones in internal organs (liver, stomach, lungs). Immunocytological identification of the heart cell type(s) expressing the virus revealed that the only cells infected were cardiomyocytes. When the constructs were inoculated at E5, no lacZ+ clones appeared in the heart but all were located in the cephalic skin. In order to examine the relationship between viral integration and expression, DNA of different organs or tissues from lacZ stained embryos was analyzed by PCR. A tight correlation between integration and expression in the heart and in the skin was revealed in most cases. In contrast, a significant PCR signal was often detected in the liver or the stomach despite weak or absent expression as revealed by lacZ+ clones.

We then investigated the influence of envelope glycoprotein subgroups on the tropism of these constructs. The lacZ vector driven by RAV-2 LTRs was packaged as subgroups A, B or E viral particles. The A subgroup, used in the part of the study described above, infects both chick and quail while the B and E subgroups are specific for chick or quail respectively. These B and E subgroups induced lacZ+ clones in the heart (after E3 injection) while no clones or only a few were detected in the skin either after E3 or E5 injection. The following conclusions can be drawn: 1) cardiomyocytes are at E3 the major target for integration and expression of ALV-derived viruses in vivo; 2) targets change rapidly with embryonic age; and 3) tissue-specific infections depend on the envelope subgroup, thus presumably on the presence of the cognate receptor. This study clearly indicates that E3 inoculation of ALV-based retroviral vectors is a simple and powerful method to transfer gene sequences into cardiomyocytes and epidermal cells.     

反转录病毒基因治疗载体及其安全性评价

反转录病毒载体是目前基因治疗中应用最广泛的基因运载工具之一,具有能稳定整合入宿主细胞、持久表达目的基因、感染细胞范围广、基因容量较大等优点,但其生物安全性还存在争议。在简要介绍反转录基因治疗病毒载体的基础上,对反转录病毒载体中外源目的基因的表达调控及反转录病毒载体的安全性评价进行了综述。     

诱发血管瘤型J亚群禽白血病病毒gp85基因的克隆与表达

2007年7月至11月,中国开产前后的商品海兰褐蛋鸡群大面积暴发血管瘤,造成巨大经济损失。将病料接种DF1细胞,通过PCR和间接免疫荧光(IFA)确定此次暴发的血管瘤为J亚群白血病病毒(ALV-J)感染引起。从患病鸡群中分离到5株ALV-J(前4株已经报道),将第5株病毒命名为WS0705。为研究该毒株抗原性的特点,用PCR方法扩增出gp85基因,并克隆进pMD18-T载体进行测序。氨基酸系统进化树分析显示WS0705与英国ALV-J原型毒株HPRS-103同源性最高。从已构建的质粒pMD18-T-WS0705gp85中酶切回收gp85基因,构建重组转移载体pFastBacH Tb-WS0705gp85。利用Bac-to-Bac表达系统获得了重组杆状病毒rBac-WS0705gp85。间接免疫荧光和Western blot检测WS0705gp85基因的表达产物。间接免疫荧光显示,构建的重组杆状病毒感染的Sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的Sf9细胞蛋白显示出约35kD的阳性条带。结果表明,WS0705gp85基因在Sf9细胞中得到良好的表达,并且其编码产物完全可以被外源性ALV-J的特异性单抗JE9识别,进一步证明本次暴发血管瘤的病原为ALV-J,并为进一步开发ALV-J相关诊断产品奠定了基础。     

Characterization of Determinants for Envelope Binding and Infection in Tva, the Subgroup A Avian Sarcoma and Leukosis Virus Receptor

Tva is the cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV-A). The viral interaction domain of Tva is determined by a 40-residue, cysteine-rich module closely related to the ligand binding domain of the human low-density lipoprotein receptor (LDLR). In this report, we examined the role of the LDLR-like module of Tva in envelope binding and viral infection by mutational analysis. We found that the entire LDLR module in Tva is essential for efficient binding to the viral envelope protein. However, the 17 N-terminal residues of this module can be deleted without affecting receptor function, suggesting that the major determinants for viral entry are located at the C terminus of the module. The effect on viral infection of many amino acid substitutions and deletions in the LDLR module is context dependent, suggesting that the residues important for viral entry are dispersed throughout the LDLR module. In addition, we found that all 27 mutations at residues D46, E47, and W48 greatly reduced envelope binding. These results are discussed in relation to a recently elucidated structure for an LDLR module.     

H9N2型禽流感病毒核蛋白的原核表达及多克隆抗体制备

目的:利用大肠杆菌表达H9N2禽流感病毒(AIV)核蛋白(NP)与GST的融合蛋白并分离纯化,进行动物免疫制备多克隆抗体。方法:根据AIV NP基因序列设计引物,将已经获得的NP基因定向克隆到GST融合原核表达载体pGEX-KG并转化大肠杆菌,在IPTG诱导下获得高效表达。经谷胱甘肽层析柱分离纯化蛋白,制备抗原免疫家兔,得到pGEX-KG-NP多克隆抗体。结果:SDS-PAGE分析显示融合表达蛋白GST-NP相对分子质量约82 000,表达量约占菌体总蛋白的20%。Western-blot和ELISA检测结果表明,重组NP能与鸡抗AIV抗体发生明显的抗原抗体反应。自制的多克隆抗体能特异地与NP相互作用,可用于AIV病原诊断。结论:获得了NP基因的高效表达产物;制备了效价和特异性良好的抗重组NP多克隆抗体。经实验验证表达产物具有活性,多克隆抗体效价高,特异性强,为AIV病原诊断试剂的研发奠定了基础。     

Avian developmental antigens: Expression on erythrocytes of chickens,Japanese quail,and quail-chicken hybrids

Monoclonal and polyclonal antibodies were used to examine the expression of three erythroid developmental antigen systems in the chicken, Japanese quail, and quail-chicken hybrid. Chicken fetal antigen (CFA), quail fetal antigen (QFA), and chicken adult antigen (CAA) each represent a series of cell-surface glycorproteins associated with the development of avian hematopoietic cells. Monoclonal anti-CFA antibodies from clones 190-4 and 288-1.1.1.2 supernatants were shown to react against epitopes associated with CFA determinants 8 and 2, respectively. Using complement-mediated microcytotoxicity, these reagents permitted the identification of different erythroid subpopulations in the neonatal chicken and hybrid; therefore, heterogeneity in cell surface CFA determinants among mature peripheral erythrocytes should serve as a useful tool for analyzing erythroid development. In the case of CAA, erythrocytes from adult hybrids were found to express the same complement of CAA determinants identified in the chicken, and CAA appeared much earlier in the hybrid than in either of the parental species. Similarly, two species-restricted fetal antigens associated with similar glycoproteins, CFA8 and QFA, had similar developmental profiles in their respective species, the chicken and quail. In contrast, these antigens were dominantly expressed but exhibited different developmental profiles on erythrocytes from the hybrids. While quail-chicken hybrids exhibited apparent genomic interactions in the expression of these developmental antigens, no evidence for the existence of hybrid-specific fetal antigens was obtained.     

流行性乙型脑炎病毒GSS株prM、E和NS1蛋白基因的克隆及在非复制型痘苗病毒中的表达

克隆流行性乙型脑炎(乙脑)病毒野毒(JEV)GSS株前膜蛋白信号序列、前膜蛋白(prM)、包膜蛋白(E)、非结构蛋白-1(NSl)和非结构蛋白NS2a的编码基因,并与非复制型痘苗病毒载体NTV进行同源重组,构建了乙脑病毒非复制型重组痘苗病毒疫苗株NTVA(E／L)JEV。通过：PCR和Southern blot检测证明,在非复制型痘苗病毒中有乙暗病毒prM信号序列、prM、E、NS1和NS2a基因的插入：Western blot检测证明,重组病毒可以在细胞内成功地表达prM、E和NSl蛋白,并可将prM、E和NSl蛋白分泌到细胞培养上清中;免疫荧光检测证明,E和NSl蛋白主要分布在细胞膜上。电镜下可见分泌到细胞外的病毒样颗粒。     

布氏鲳鲹GHRH基因克隆、组织和胚胎表达模式

促生长激素释放激素(growth hormone releasing hormone, GHRH)主要生物学功能是刺激垂体细胞分泌生长激素,已被证实是动物体生长轴的重要调控因子之一,布氏鲳鲹是一种生长快速的海洋鱼类,为了揭示其代谢旺盛的调节机制,本研究从GHRH入手,利用RACE技术和qPCR方法对布氏鲳鲹GHRH基因进行了克隆、组织和胚胎表达模式研究。实验结果显示,布氏鲳鲹GHRH基因cDNA序列全长1019bp,5’UTR、3’UTR长度分别为327 bp和164 bp,开放阅读框528 bp,共编码175个氨基酸;同源性分析结果表明,布氏鲳鲹GHRH基因与其它鲈形目鱼类的同源性在91%以上。布氏鲳鲹GHRH基因的表达区域大多都集中在中枢系统,其中下丘脑表达量最高;GHRH在受精卵期到后续发育过程中均检测到表达,其表达水平在仔鱼期达到最高。序列分析、组织及胚胎表达的结果表明,布氏鲳鲹GHRH的调节模式仍然可能通过下丘脑调节垂体释放GH,GHRH在个体发育的较早阶段即开始发挥作用。本研究掌握了布氏鲳鲹GHRH基因的基本规律,为进一步研究生长轴的调控提供了理论参考。     

鸡传染性支气管炎病毒S1基因与猪IgG Fc基因在HeLa细胞中的融合表达

根据Genbank中发表的猪IgG Fc段基因及IBV S1基因序列,设计并合成引物。以猪肝组织总RNA为模扩增出猪IgG Fc基因,以含全长IBV M41 S基因的质粒为模板扩增出IBV S1基因,分别克隆至T载体。DNA测序表明,所获得的IBV S1基因大小为1.5 kb,IgG Fc大小为1kb,序列正确。将IBV S1与IgG Fc基因串连,插入含有人组织型纤维蛋白溶酶原激活物分泌信号肽序列(tPA)真核表达载体pcDNA3.1-tPA上,在HeLa细胞上进行瞬时融合表达。经免疫荧光和斑点杂交检测,表达产物同时具有IBV S1蛋白和IgG Fc活性。     

Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin genes

Expression vectors for cDNA of the κ and λ1 chains of a monoclonal antibody directed against creatine kinase were introduced into murine myeloma cells. κ and γ1 cDNA were either under the control of the SV40 early promoter or of the cognate promoters and enhancers of the light- and heavy-chain genes. Secretion of immuno-reactive κ and γ1 chains into the culture medium was demonstrated with the SV40 promoter as well as with the cognate promoters. Expression of y 1 cDNA with the SV40 early promoter was about twice as high as with the heavy-chain promoter and enhancer. Expression of κ cDNA under the control of the S V40 early promoter was about 17 times higher than with the light-chain promoter and enhancer. These expression levels were compared to those of a genomic immunoglobulin (Ig) κ determinant, including introns. Such an entire κ gene led to expression of the light chain at levels double those with the κ cDNA construction using the SV40 promoter and about 35 times as high when using κ cDNA and the cognate promoter and enhancer. This result might indicate that, besides the cognate promoter and enhancer elements, other intragenic elements are involved in the regulation of Ig expression. However, the SV40 early promoter seems to be able to compensate for the absence of these postulated regulatory elements probably located in the introns.     

Littermate Influence on Infant Growth in Mice: Comparison of SJL/J and ICR as Cotransferred Carrier Embryos

In mice, a minimum number of healthy embryos is required to trigger and maintain pregnancy. Therefore, when recovering mouse embryos from a limited litter, one useful technique is to transfer carrier ICR embryos along with the embryos of interest, a technique referred to as cotransfer. In this study, we examined suitable mouse strains for cotransfer with C57BL/6J (B6) embryos in regards to the maintenance of pregnancy, number of pups born, intrauterine growth, and postnatal growth. Because the coat color of B6 is black, we compared two white coat-colored strains, SJL/J and ICR. Cotransfer of SJL/J and ICR embryos had similar effects on maintenance of pregnancy, number of pups born, and intrauterine growth. However, the postnatal growth of B6 mouse pups cotransferred and grown with SJL/J pups was better than for B6 mouse pups cotransferred and grown with ICR pups, suggesting competition among littermates. These results demonstrate that cotransfer of SJL/J embryos will be useful not only as carrier embryos with B6-background embryos but also as a model system to examine littermate competition.     

玉米杂种与亲本穗分化期功能叶基因差异表达与杂种优势

为探讨玉米杂种优势的分子机理,以10个玉米自交系及其组配的38个杂交种为材料,利用cDNA-AFLP技术,分析杂种与亲本在玉米雌穗小穗分化期功能叶片的基因差异表达类型与主要农艺性状的杂种表现及杂种优势的关系。研究表明：（1）杂种的基因相对于其双亲,存在质和量的表达差异,其中质的差异表达类型包括：单亲沉默表达,双亲沉默表达,亲本显性表达和杂种特异表达等类型。（2）在雌穗小穗分化期,同一差异表达类型中不同杂交组合间差异很大;从总体平均看,杂种特异表达类型占25.22%,亲本显性表达类型占21.46%,双亲沉默表达类型占8.27%,单亲沉默表达类型占33.49%。（3）单亲沉默表达与株高的杂种表现呈显著正相关;双亲沉默表达与穗粗的杂种优势呈显著负相关,显性表达与行粒数和单株粒重的杂种优势呈显著负相关,其余表达类型与所有农艺性状杂种表现及杂种优势均不相关,并对结果进行了讨论。     

Supramolecular assemblies of zwitterionic nanoliposome-polynucleotide complexes as gene transfer vectors: Nanolipoplex formulation and in vitro characterisation

Synthetic gene transfer vectors based on zwitterionic nanoliposome-DNA assemblies (nanolipoplexes), formed by the mediation of magnesium ions, were prepared by a scalable method without employing volatile solvents, high-shear force treatments or extrusion. The zwitterionic nanolipoplexes (NLP) were formulated with PC (phosphatidylcholine) and DPPC (a natural lung surfactant) incorporating different amounts of cholesterol (CHOL). The resulting structures were characterised in terms of their morphology, size and DNA content. In addition, the toxicity and transfection efficiency of the nanolipoplexes were evaluated in cultured Chinese hamster ovary-K1 (CHO-K1) cells. The effects of the multivalent cation Mg²⁺ on nanoliposome-DNA transfection potency were evaluated. Formulations containing 10% CHOL showed maximum transfection efficiency and the optimum amount of Mg²⁺ ions for transfection with minimum cytotoxicity was ca. 20 mM. The zwitterionic formulations showed significantly less cytotoxicity compared to a commercially available cationic liposome reagent or polyethylenimine (PEI) while they were superior in terms of gene transfer potency. The zwitterionic vectors formulated in this study avoid the use of toxic cationic lipids as well as toxic solvents and may have potential application in gene therapy. The new method will enable scale-up and manufacture of safe and efficacious transfection vehicles required for preclinical and clinical studies. Based on the advantages and superiority of the formulated nanolipoplexes, this method allows for the acceleration of nanolipoplex formulation, enabling the rapid development and evaluation of novel carrier systems for genes and other drugs.     

Forty years of embryo transfer in cattle: A review focusing on the journal Theriogenology, the growth of the industry in North America,and personal reminisces

After the first successful transfer of mammalian embryos in 1890, it was approximately 60 years before significant progress was reported in the basic technology of embryo transfer (ET) in cattle. Starting in the early 1970s, technology had progressed sufficiently to support the founding of commercial ET programs in several countries. Today, well-established and reliable techniques involving superovulation, embryo recovery and transfer, cryopreservation, and IVF are utilized worldwide in hundreds, if not thousands, of commercial businesses located in many countries. The mean number of embryos produced via superovulation has changed little in 40 years, but there have been improvements in synchrony and hormonal protocols. Cryopreservation of in vivo-derived embryos is a reliable procedure, but improvements are needed for biopsied and in vitro-derived embryos. High pregnancy rates are achieved when good quality embryos are transferred into suitable recipients and low pregnancy rates are often owing to problems in recipient management and not technology per se. In the future, unanticipated disease outbreaks and the ever-changing economics of cattle and milk prices will continue to influence the ET industry. The issue of abnormal pregnancies involving in vitro embryos has not been satisfactorily resolved and the involvement of abnormal epigenetics associate with this technology merits continued research. Last, genomic testing of bovine embryos is likely to be available in the foreseeable future. This may markedly decrease the number of embryos that are actually transferred and stimulate the evolution of more sophisticated ET businesses.     

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non-polarized and polarized cells

BACKGROUND: Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. METHODS: After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively. RESULTS: PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency. CONCLUSIONS: We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes.     

Methylmercury in Marine Ecosystems: Spatial Patterns and Processes of Production,Bioaccumulation, and Biomagnification

The spatial variation of MeHg production, bioaccumulation, and biomagnification in marine food webs is poorly characterized but critical to understanding the links between sources and higher trophic levels, such as fish that are ultimately vectors of human and wildlife exposure. This article discusses both large and local scale processes controlling Hg supply, methylation, bioaccumulation, and transfer in marine ecosystems. While global estimates of Hg supply suggest important open ocean reservoirs of MeHg, only coastal processes and food webs are known sources of MeHg production, bioaccumulation, and bioadvection. The patterns observed to date suggest that not all sources and biotic receptors are spatially linked, and that physical and ecological processes are important in transferring MeHg from source regions to bioaccumulation in marine food webs and from lower to higher trophic levels.     

猪生殖和呼吸综合征病毒B13株ORF7基因的克隆及在杆状病毒系统中的表达

利用单管RT－PCR方法扩增猪生殖和呼吸综合征病毒（PRRSV）分离株B13株包括ORF7基因的片段,并对其序列进行了测定,结果PRRSV分离株B13ORF7基因长度为384bp,编码128个氨基酸组成的15kD蛋白。与已发表的PRRSV LV株、VR－2332株进行同源性比较,发现核苷酸同源性分别为99.2%、59.4%;氨基酸同源性分别为98.4%、54.7%。。表明PRRSV分离株B13在基因结构上可能与LV株同属于欧洲亚群。同时构建了重组转移载体质粒pAcGHLT-B-ORF7,且该重组转移载体质粒与线性化苜蓿丫纹夜蛾核型多角体病毒（AcMNPV-SVI^-G)基因组DNA（baculo gold linearized baculovirus DNA）共转染草地夜蛾（Spodoptcra frugiperda,Sf9）细胞,得到重组病毒AcMNPV-OCC^--GST-6xHis-ORF7。在感染了重组病毒的Sf9细胞中检测到分子量为46kD的ORF7基因的GST融合蛋白表达产物,能被猪抗PRRSVB13株多克隆血清所特异识别。此结果为PRRS新型诊断抗原的研制奠定了基础。     

Characterization of Growth and Reproduction Performance,Transgene Integration,Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer

Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.