Construction and expression of a recombinant adeno-associated virus that harbors a human beta-globin-encoding cDNA.

Towards a goal of using recombinant adeno-associated viruses (AAV) for the gene therapy of hemoglobinopathies we had previously constructed plasmid pAV h beta G psi 1, which contained a human beta-globin-encoding cDNA (HBB) downstream from the P40 promoter of AAV2 DNA [Ohi et al., Gene 89 (1990) 279-282]. Transfection of the plasmid into human 293 cells (embryonal kidney cell line) resulted in the expression of HBB at the mRNA level as well as rescue and replication of the recombinant AAV genome (Ohi et al., ibid.). The present study demonstrates that the replicated recombinant DNA was packaged into an intact virion by transcomplementation with pAV2 or the defective helpers, pAV delta Bam or pAVXB. The recombinant virus could be isolated by equilibrium CsCl density gradient, the density of which was about 1.4 g/cm3. The defective helpers are used to produce wild-type AAV-free recombinant AAV. The recombinant AAV were infectious and expressed chimeric mRNAs containing the HBB sequence in virus-infected 293, KB (oral epidermoid carcinoma cell line) and K562 (human erythroleukemia cell line) cells. The importance of the infectivity and expression of the recombinant AAV in hematopoietic cells is discussed in the context of gene therapy of hemoglobinopathies.     

Introduction of sequences encoding functional human adenosine deaminase into mouse cells using a retroviral shuttle system

A retroviral packaging system was used to generate a murine virus carrying sequences encoding human adenosine deaminase (ADA). To this end, human ADA cDNA was inserted into the retroviral shuttle vector pZIP-NeoSV(X)1. This vector provides all of the cis-acting sequences necessary for the efficient packaging and transmission of the viral genome as well as a selectable gene for G418 resistance. Transfection of this recombinant plasmid into cells that provide essential virus products (psi-2 cells) yielded cell lines that stably produced virions carrying the coding sequence of human ADA. We have used these virions to infect NIH3T3 cells, which after 48 h synthesized catalytically active human ADA. Furthermore, G418-resistant cell lines were obtained from the virus-infected NIH3T3 cells that stably produced the human ADA enzyme.     

FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

Molecular structures of human argininosuccinate synthetase pseudogenes. Evolutionary and mechanistic implications

In the human genome there is one expressed gene for argininosuccinate synthetase and 14 pseudogenes. A cDNA coding for human argininosuccinate synthetase was used to screen a human genomic library. Twenty-five unique genomic clones were isolated and extensively characterized. At least seven clones represented processed argininosuccinate synthetase pseudogenes that lost the introns in the expressed gene. Restriction mapping demonstrated that these processed pseudogenes were located in distinct regions of the human genome. Complete nucleotide sequences of two processed pseudogenes, psi AS-1 and psi AS-3, and a partial sequence of psi AS-7 were determined. Both psi AS-1 and psi AS-3 had an adenine-rich region at their 3' end and were flanked by distinct imperfect direct repeats. A comparison of these pseudogene sequences to that of the cDNA demonstrated that psi AS-1 and psi AS-3 were 93% homologous to the cDNA, whereas psi AS-7 was 89% homologous to the cDNA. Therefore, it is estimated that psi AS-1 and psi AS-3 were created 10-11 million years ago, whereas psi AS-7 arose approximately 21 million years ago. We have estimated the evolutionary rate for the expressed argininosuccinate synthetase gene based on the sequences of psi AS-1 and psi AS-3. These data indicate that the expressed argininosuccinate synthetase gene is evolving at a rate similar to that of the beta-globin gene and much faster than the alpha-tubulin gene. Furthermore, a comparison of the sequences of psi AS-1 and psi AS-3 suggests the possibility that these pseudogenes arose from a common intermediate.     

Effect of butyrate on the expression of the human preprourokinase gene introduced into Chinese hamster ovary cells

A plasmid containing the human preprourokinase gene cDNA under the control of the simian virus 40 early region promoter was introduced into CHO-K1 cells and recombinant cell lines secreting a relatively high level of urokinase were obtained. In the course of studying the effects of various agents on the recombinant cell lines, we found that exposure of recombinant cells to 5 mM butyrate for 24 hours resulted in a 2-3 fold increase in urokinase production. The induction by butyrate was dose-dependent. The half maximal dose was approximately 2 mM; maximal stimulation occurred at 5-10 mM. Cell growth, on the other hand, was inhibited by butyrate concentrations greater than 2.5 mM. The response of cells to butyrate was rapid: a significant increase in urokinase production was observed 6 hours after exposure to 5 mM butyrate. Butyrate treatment increased not only the extracellular level but also the intracellular level of urokinase.     

Infection of human cells by murine ecotropic viruses: retroviral vectors carrying the hygromycin resistance-encoding gene

The construction of a new retroviral vector, pSKV, is described. This vector carries two unique cloning sites, located between two Moloney leukemia virus-derived LTR, into which genes of interest may be introduced. The gene encoding hygromycin resistance (Hy^R) was subsequently introduced into one of the two sites, producing a second vector (pSKV/Hy^R) containing a unique SfiI site for the introduction of cDNA clones under the control of the cytomegalovirus (CMV) promoter (P-CMV). The cDNA (mH13), encoding a protein that has been shown to serve as a murine ecotropic retroviral receptor in transient assays, was cloned into the SfiI site (pSKV/Hy^R/mH13). Both constructs can be packaged into retroviral particles following transfection into an appropriate packaging cell line.

Stable transfectants of the human glioblastoma cell line (U118MG) carrying each of these two constructs were generated by transfection and subsequent Hy selection. Clones expressing both the selectable marker and the mH13 gene, but not those expressing only the selectable marker, are shown to be susceptible to infection with murine ecotropic retroviral particles. These cells (Hy^R and mH13 positive) were then exposed to CRE/Xtk culture supernatant, a packaging cell line producing ecotropic retroviral particles carrying the HSV-TK (Herpes simplex virus-thymidine kinase) and neo^R(neomycin-resistance) genes. Selection was in the presence of G418. In vitro growth of the U118MG/Hy^R/mH13/TK cells, but not that of the U118MG/HyR/mH13 cells, was inhibited by ganciclovir (GCV), indicating the successful transfer of HSV-TK by infection of human cells with murine retroviruses via the mH13 product.     

hEPO cDNA重组逆转录病毒的构建

通过DNA重组技术,将不含非编码区的hEPO cDNA片段重组到逆转录病毒质粒pLXSN, pLNCX中重组质粒转染PA317细胞后,经G418筛选,抗性克隆细胞培养上清能成功地感染NIH3T3细胞,使之在筛选培养基中形成典型的G418抗性克隆,该克隆细胞染色体中成功地整合了EPOcDNA,并且表达出有生物学活性的红细胞生成素(EPO)产物。     

In vitro synthesis of infectious retroviral RNA.

A plasmid was constructed in which a T7 RNA polymerase promoter was placed upstream of a recombinant amphotropic retrovirus genome containing a selectable neomycin resistance gene. To test the infectivity of the RNA produced by T7 RNA polymerase in vitro, the RNA was microinjected into the nuclei of psi 2 packaging cells. Infectious particles conferring G418 resistance were released.     

Expression of the hepatitis B virus surface,core and E antigen genes by stable rat and mouse cell lines

A recombinant plasmid containing four tandem head-to-tail copies of the HBV⁴ genome has been constructed and introduced into thymidine kinase-deficient mouse and rat fibroblast cells by co-transformation with the thymidine kinase gene from Herpes simplex virus as a selectable marker. Several mouse cell lines that synthesize HBsAg and four lines (three mouse and one rat) that synthesize both HBsAg and HBeAg were isolated. The rat line and at least one of the L cell lines that synthesizes HBeAg also produce small quantities of HBcAg. The rat line has many partial and 18 to 20 complete copies of the HBV genome integrated into high molecular weight DNA, whereas the L cells that produce HBeAg have only two to three complete copies of HBV genome. The kinetics of synthesis of HBsAg and HBeAg by the rat line are similar to that for HBsAg synthesis by two human hepatoma lines, and the HBsAg secreted by this line has similar biophysical characteristics to that found in human serum. The results suggest that the configuration of the recombinant molecule used may be an important prerequisite for the expression of HBcAg and HBeAg, and we speculate that the biogenesis of their messenger RNA may proceed via a precursor that is greater than the length of the HBV genome.     

逆转录病毒介导的人血小板生成素(TPO)在骨髓基质细胞中的表达

为探讨逆转录病毒介导的TPO基因在人骨髓基质细胞系HFCL中的表达,利用脂质体法将含TPO基因的逆转录病毒载体导入HF-CL细胞中,RT-PCR和基因组DNAPCR分析证实mRNA水平有表达,基因组中整合有Neo基因和TPO基因。TPO依赖细胞株TD-3检测生物学活性表明转染的骨髓基质细胞分泌TPO。上述结果为进一步研究转基因骨髓基质细胞对造血细胞的调控作用提供了必要的基础资料。     

人神经元素3基因重组逆转录病毒表达载体的构建及其包装细胞株的建立

为构建表达人神经元素3基因(neurogenin 3,ngn3)的重组逆转录病毒载体,建立稳定表达ngn3的包装细胞株,本研究以流产人胎儿胰腺组织为材料,通过RT-PCR方法克隆出人ngn3基因,将其连接到pMD18-T载体上并测序,结果表明,测序得到的基因序列与发表的人ngn3基因序列(GenBank Accession No.BC126468)完全一致。将EcoRI和HpaI双酶切后的基因片段构建到pMSCV-neo逆转录病毒载体中,酶切鉴定结果表明,pMSCV-ngn3重组逆转录病毒载体构建成功。脂质体法将pMSCV-ngn3重组载体导入PT67包装细胞,G418筛选后,对得到的细胞株进行RT-PCR和免疫组化检测,结果显示,该细胞株在mRNA水平和蛋白水平均稳定表达Ngn3;收集该细胞株的培养上清液,进行RT-PCR检测及电镜观察,结果表明,该细胞株将导入的重组逆转录病毒载体pMSCV-ngn3包装成了具有感染性的病毒颗粒,并将其释放到了培养上清液中。以上结果表明PT67-ngn3包装细胞株建立成功。该细胞株的成功建立,为下一步将ngn3基因应用于提高人胎儿胰腺祖细胞诱导分化效率方面的研究奠定了基础。     

人神经营养因子-3重组逆转录病毒的包装与鉴定

在逆转录病毒载体pLXSN中引入人神经营养因子-3(hNT-3),构建成重组质粒pLXSN-NT3,转染包装细胞PA317后经G418筛选得到几个稳定产毒的细胞克隆.测定这几个克隆的病毒滴度,最高达到1.60×105,且产毒细胞株不产生复制型病毒.PCR和大乳鼠背根神经节活性检测证明hNT-3已整合到宿主细胞基因组并能稳定表达.     

Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes.

Recombinant retroviruses containing the trans activator genes of human T-cell leukemia virus (HTLV) type II and human T-cell lymphotropic virus type III were constructed. The trans activator genes tat II and tat III were inserted into the murine retroviral vector pZIPNEOSV(X)1. Recombinant plasmids were transfected into the psi 2 and psi AM packaging cell lines that produce murine leukemia virions containing no retroviral RNA. Functional tat II and tat III gene products were expressed as demonstrated by trans activation of HTLV type I and II and human T-cell lymphotropic virus type III long terminal repeat-directed gene expression in the respective infected cells. Use of these recombinant vectors permits high-efficiency gene transfer into a wide variety of cells, thereby providing the opportunity to study the biochemical effects associated with tat II and tat III gene expression.     

Identification of three human pseudogenes for subunit VIb of cytochrome c oxidase: a molecular record of gene evolution.

Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned human cDNA coding for COX subunit VIb. The nucleotide sequences of the pseudogenes, designated psi COX6b-1, psi COX6b-2 and psi COX6b-3, were determined. Pseudogene psi COX6b-1 bears all the hallmarks of a processed pseudogene and diverged from the parental gene after the divergence of man and cow. Alu repetitive elements were integrated into the structural sequences of the other two pseudogenes. Comparison with the human and bovine cDNA sequences encoding COX subunit VIb suggests that psi COX6b-2 and psi COX6b-3 were formed earlier in evolution than psi COX6b-1. Genomic Southern analysis indicated that a few more pseudogenes for COX subunit VIb are likely to be present in the human genome. Identical nt differences with respect to the human cDNA sequence in the pseudogenes provide some clues on the evolution of the ancestral gene coding for COX subunit VIb.     

Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder.

A high-titer, recombinant retroviral vector produced in psi 2 packaging cells has been used to introduce the murine interleukin-3 (IL-3) gene into mouse hematopoietic cells. Integration and expression of the IL-3 gene was observed in spleen foci from which could be derived factor-independent, continuously proliferating cell lines. Irradiated or genetically anemic W/Wv recipients of infected hematopoietic cells developed a myeloproliferative syndrome characterized by a marked elevation in leukocyte count, bone marrow hyperplasia, and enlargement of the liver and spleen. The syndrome reflected proliferation of one or more stem cell clones, the progeny of which were capable of repopulating secondary recipients. One animal developed the syndrome primarily by a paracrine mechanism. Endogenous IL-3 production caused amplification of hematopoietic cells but did not appear to alter the maturational or self-renewal potential of these cells.     

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.     

人c-myc转基因细胞的建立

目的:建立人c-myc转基因细胞。方法:通过成功构建c-myc逆转录病毒表达载体,并经脂质体介导转染包装细胞293T,收集产重组病毒的293T培养上清,运用NIH3T3细胞测定了病毒滴度,用适当浓度的病毒感染L929细胞,经用Zeocin选择性培养基筛选细胞。结果:得到稳定高表达c-myc基因的L929转基因细胞。结论:运用逆转录病毒转染法可得到高表达的转基因细胞。