Transfection Of Genomic DNA Research Articles

Identification of cGAS as an innate immune sensor of extracellular bacterium Pseudomonas aeruginosa.

SummaryCyclic GMP-AMP synthase (cGAS) is reported essential for detecting intracellular bacteria. However, it remains to be determined whether and how cGAS is involved in extracellular bacterial infection. Here, we report that cGAS is essential for mediating type I interferon (IFN) production in infection by multiple extracellular pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. In addition, the canonical cGAS-stimulator of interferon gene (STING)-IFN axis is required for protecting mice from P. aeruginosa-induced mouse acute pulmonary infection, confirmed in cGAS pathway-specific gene deficiency mouse models. cGAS−/− and STING−/− mice exhibited reduced type I IFNs production, excessive inflammatory response accompanied with decreased resistance to P. aeruginosa challenge. Unfolded protein response was also modulated by cGAS through IRF3 and type I IFNs under P. aeruginosa infection. Collectively, these findings uncover the importance of cGAS in initiating immune responses against extracellular bacterial infection.

Herceptin: A First Assault on Oncogenes that Launched a Revolution

This year's Lasker Clinical Research Award honors H. Michael Shepard, Dennis J. Slamon, and Axel Ullrich for their invention of Herceptin, the first monoclonal antibody that blocks a cancer-causing protein, and for its development as a life-saving therapy for women with breast cancer.

Pattern recognition receptor-initiated innate antiviral responses in mouse epididymal epithelial cells.

Viral infections of the epididymis may impair male fertility and spread sexually transmitted pathogens. The innate antiviral immune responses in the epididymis have yet to be intensively investigated. This study found that mouse epididymal epithelial cells (EECs) constitutively express several viral sensors, including TLR3, retinoic acid-inducible gene I, and DNA-dependent activator of IFN regulatory factors. Other DNA sensors, including p204 and cGMP-AMP synthase, can be induced by transfection of synthetic HSV genomic DNA (HSV60). TLR3 and retinoic acid-inducible gene I in EECs can be activated by their common agonist, polyinosinic-polycytidylic acid [poly(I:C)]. The signaling pathway of DNA sensors can be initiated by HSV60. Both poly(I:C) and HSV60 induced the expression of type 1 IFNs and various antiviral proteins, including IFN-stimulated gene 15, 2',5'-oligoadenylate synthetase, and myxovirus resistance 1. Poly(I:C), but not HSV60, also dramatically induced the expression of major proinflammatory cytokines, including TNF-α and MCP-1, in EECs. In vivo assay confirmed that the local injection of poly(I:C) or HSV60 induced the innate antiviral responses in EECs. This study provided novel insights into the mechanisms underlying the innate antiviral responses in the mouse epididymis.

A temporal gate for viral enhancers to co-opt Toll-like-receptor transcriptional activation pathways upon acute infection.

Viral engagement with macrophages activates Toll-Like-Receptors (TLRs) and viruses must contend with the ensuing inflammatory responses to successfully complete their replication cycle. To date, known counter-strategies involve the use of viral-encoded proteins that often employ mimicry mechanisms to block or redirect the host response to benefit the virus. Whether viral regulatory DNA sequences provide an opportunistic strategy by which viral enhancer elements functionally mimic innate immune enhancers is unknown. Here we find that host innate immune genes and the prototypical viral enhancer of cytomegalovirus (CMV) have comparable expression kinetics, and positively respond to common TLR agonists. In macrophages but not fibroblasts we show that activation of NFκB at immediate-early times of infection is independent of virion-associated protein, M45. We find upon virus infection or transfection of viral genomic DNA the TLR-agonist treatment results in significant enhancement of the virus transcription-replication cycle. In macrophage time-course infection experiments we demonstrate that TLR-agonist stimulation of the viral enhancer and replication cycle is strictly delimited by a temporal gate with a determined half-maximal time for enhancer-activation of 6 h; after which TLR-activation blocks the viral transcription-replication cycle. By performing a systematic siRNA screen of 149 innate immune regulatory factors we identify not only anticipated anti-viral and pro-viral contributions but also new factors involved in the CMV transcription-replication cycle. We identify a central convergent NFκB-SP1-RXR-IRF axis downstream of TLR-signalling. Activation of the RXR component potentiated direct and indirect TLR-induced activation of CMV transcription-replication cycle; whereas chromatin binding experiments using wild-type and enhancer-deletion virus revealed IRF3 and 5 as new pro-viral host transcription factor interactions with the CMV enhancer in macrophages. In a series of pharmacologic, siRNA and genetic loss-of-function experiments we determined that signalling mediated by the TLR-adaptor protein MyD88 plays a vital role for governing the inflammatory activation of the CMV enhancer in macrophages. Downstream TLR-regulated transcription factor binding motif disruption for NFκB, AP1 and CREB/ATF in the CMV enhancer demonstrated the requirement of these inflammatory signal-regulated elements in driving viral gene expression and growth in cells as well as in primary infection of neonatal mice. Thus, this study shows that the prototypical CMV enhancer, in a restricted time-gated manner, co-opts through DNA regulatory mimicry elements, innate-immune transcription factors to drive viral expression and replication in the face of on-going pro-inflammatory antiviral responses in vitro and in vivo and; suggests an unexpected role for inflammation in promoting acute infection and has important future implications for regulating latency.

Triptolide inhibits human immunodeficiency virus type 1 replication by promoting proteasomal degradation of Tat protein.

BackgroundPlants remain an important source of new drugs, new leads and new chemical entities. Triptolide is a diterpenoid epoxide isolated from Tripterygium wilfordii Hook F that possesses a broad range of bioactivities, including anti-inflammatory, immunosuppressive and anti-tumor properties. The antiviral activity of triptolide against human immunodeficiency virus type 1 (HIV-1) has not been reported.ResultsIn this study, nanomolar concentrations of triptolide were shown to potently inhibit HIV-1 replication in vitro. To identify the step(s) of the HIV-1 replication cycle affected by triptolide, time-of-addition studies, PCR analysis and direct transfection of viral genomic DNA were performed. The results of these experiments indicated that triptolide acts at the stage of viral gene transcription. In addition, a luciferase-based reporter assay that allows quantitative analysis of long terminal repeat (LTR)-driven transcription showed that Tat-induced LTR activation was impaired in the presence of triptolide. Moreover, Western blot analysis of exogenous gene expression (driven by the human elongation factor 1 α subunit promoter) in transiently transfected cells revealed that triptolide specifically reduces the steady-state level of Tat protein, without suppressing global gene expression. Further studies showed that triptolide accelerates Tat protein degradation, which can be rescued by administration of the proteasome inhibitor MG132. Mutation analysis revealed that N-terminal domains of Tat protein and nuclear localization are required for triptolide to reduce steady-state level of Tat.ConclusionThis study suggests for the first time that triptolide exerts its anti-HIV-1 activity by specifically prompting the degradation of the virally encoded Tat protein, which is a novel mechanism of action for an anti-HIV-1 compound. This compound may serve as a starting point for developing a novel HIV-1 therapeutic approach or as a basic research tool for interrogating events during viral replication.

Triptolide inhibits human immunodeficiency virus type 1 replication by promoting proteasomal degradation of Tat protein

Plants remain an important source of new drugs, new leads and new chemical entities. Triptolide is a diterpenoid epoxide isolated from Tripterygium wilfordii Hook F that possesses a broad range of bioactivities, including anti-inflammatory, immunosuppressive and anti-tumor properties. The antiviral activity of triptolide against human immunodeficiency virus type 1 (HIV-1) has not been reported. In this study, nanomolar concentrations of triptolide were shown to potently inhibit HIV-1 replication in vitro. To identify the step(s) of the HIV-1 replication cycle affected by triptolide, time-of-addition studies, PCR analysis and direct transfection of viral genomic DNA were performed. The results of these experiments indicated that triptolide acts at the stage of viral gene transcription. In addition, a luciferase-based reporter assay that allows quantitative analysis of long terminal repeat (LTR)-driven transcription showed that Tat-induced LTR activation was impaired in the presence of triptolide. Moreover, Western blot analysis of exogenous gene expression (driven by the human elongation factor 1 α subunit promoter) in transiently transfected cells revealed that triptolide specifically reduces the steady-state level of Tat protein, without suppressing global gene expression. Further studies showed that triptolide accelerates Tat protein degradation, which can be rescued by administration of the proteasome inhibitor MG132. Mutation analysis revealed that N-terminal domains of Tat protein and nuclear localization are required for triptolide to reduce steady-state level of Tat. This study suggests for the first time that triptolide exerts its anti-HIV-1 activity by specifically prompting the degradation of the virally encoded Tat protein, which is a novel mechanism of action for an anti-HIV-1 compound. This compound may serve as a starting point for developing a novel HIV-1 therapeutic approach or as a basic research tool for interrogating events during viral replication.

Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.

The Study on Poly-Lysine-Alginate Microcapsules Mediated Virus Genomic DNA Transfection

The goal in this paper is to investigate the efficiency of poly-PLL (Poly-L-lysine)/ Alg (Alginate) vector mediated virus genomic DNA transfection and the virus genomic DNA’s biological activity in vivo. After Pseudorabies virus (PRV) genomic DNA being adhered to the porous СаСО3 particles, PLL and Alg were alternately polymerized on the surface of the porous DNA-СаСО3 particles to 7 layers, which were later dissolved them by EDTA to remove СаСО3 cores; the vectors in which the DNA were coated by poly-PLL/Alg, were harvested to infect the rabbits and observe the replication of viral DNA. Porous СаСО3 particles, which were obtained from the reaction between Na2CO3 and CaCl2, had an efficiency of absorbing DNA 1 µg/mg СаСО3 particles. After being coated by PLL/Alg, microcapsules were obtained with the diameter of 2-4 µm. 10.0 µg of poly-PLL/Alg-PRV DNA microcapsules could cause rabbits’ death by intramuscular injection. The identification of PCR shows that the death was caused by PRV infection. The results indicate that Poly-PLL/Alg microcapsules can mediate efficient transfection of DNA.

Molecular genetics of metastasis.

We have adopted various approaches to identifying the genes(s) involved in metastasis. The first has been to observe whether introducing a defined activated oncogene (c-Ha-ras 1) into non-neoplastic cells confers not only tumorigenicity but other characteristics of malignancy. A second approach involves transfection of total genomic DNA from highly metastatic into nonmetastatic tumour cells. Thirdly, we are studying whether treatment of weakly metastatic tumour cells with agents known to influence tumour progression and gene expression (e.g. 12-O-tetradecanoylphorbol-13-acetate or 2'-deoxy-5-azacytidine) can affect metastatic capability. It was found that 3T3 fibroblasts which incorporated and expressed the activated rasH oncogene became tumorigenic and capable of lung colonization but not spontaneously metastatic. Additionally, transfection of inert tumour cells with DNA from highly metastatic human and animal cell lines sometimes markedly augmented their spontaneous metastatic capability and their lung colony-forming potential and induced them to form deposits in many extrapulmonary sites. Treatment of some tumour cell lines with azacytidine and 12-O-tetradecanoylphorbol-13-acetate markedly increased their metastatic behaviour after subcutaneous inoculation. Because several cell divisions occurred to produce the subcutaneous tumour before the cells disseminated, we consider the changed phenotype to be heritable and probably caused by alterations in gene expression. These results suggest that components of the metastatic phenotype are heritable and highly conserved in evolution and can be conferred on previously non-metastatic tumour cells by transfer of genomic DNA. In other studies we found that metastasizing tumour cells reach all organs in the body within minutes of entry into the blood but that the distribution of subsequent secondary tumours is neither uniform nor proportional to the numbers of cells retained in each site. The patterns of distribution of metastases tend to be related to the tissue of origin of the primary tumour. This was confirmed in observations on patients with intractable malignant ascites treated with peritoneo-venous shunts. Co-culture of tumour cells with fragments of various organs in vitro supported the conclusion that the normal cells of organs can support or inhibit secondary tumour formation. These observations collectively indicate that metastasis results from acquired abnormalities in gene regulation in tumour cells, but that the resulting abnormal cell behaviour can sometimes be modified or inhibited by local or systemic conditions in the host.

Selective Translational Repression of Truncated Proteins from Frameshift Mutation-Derived mRNAs in Tumors

Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs). Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD) system. However, PTCs located within 50–55 nucleotides of the last exon–exon junction are not recognized by NMD (NMD-irrelevant), and some PTC-containing mRNAs can escape from the NMD system (NMD-escape). We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.

Molecular characterization of the largest and smallest genome segments, S1 and S12, of Rice gall dwarf virus

The sequence of the alpha-transinducing factor (alpha-TIF) of canine herpesvirus (CHV-l) was determined. Alignment of the predicted CHV-1 alpha-TIF amino acid sequence with other alpha-TIF homologues reveals a core region of similarity with divergent amino and carboxyl termini. Analysis of the CHV-1 infected cell protein 4 promoter region identified a region containing nine copies of a 52 bp repeat that showed significant up-regulation of transcription by alpha-TIF. This region contained an imperfect 'TAATGARAT' motif, the binding site for herpes simplex virus 1 alpha-TIF, with an imperfect Oct-1 binding site immediately following. The infectious laryngotracheitis virus alpha-TIF was also shown to up-regulate transcription through this region of the promoter. Transfection of CHV-1 genomic DNA failed to yield infectious virus in canine kidney cell lines. Co-transfection of genomic DNA and an alpha-TIF expression plasmid resulted in virus plaques, indicating a potential essential role for alpha-TIF in CHV-1 infection.

Promoter activity of left inverted terminal repeat and downstream sequences of porcine adenovirus type 3

Early region 1 (E1) of porcine adenovirus type 3 (PAdV-3) consists of E1A and E1B transcription units. The authentic promoter region of E1A contains a TATA box at nucleotide position (nt) 449 and a bifunctional regulatory element between nt 374 and 431, which enhances the transcription of E1A, but represses that of E1B. Here, we investigated the role of the left inverted terminal repeat (ITR) and its downstream sequences (between nt 151 and 312) in the transcription of early viral genes, and viral replication. Mutant PAdV-3s without the authentic E1A promoter region could be rescued by transfection of mutant genomic DNA into fetal porcine retina cells. Moreover, the mutant PAdV-3s produced E1A-specific mRNA and remained viable in swine testis (ST) cells suggesting that the left-terminal 151 bp including the ITR, can serve as a promoter for E1A expression. However, mutant PAdV-3s containing deletion including authentic E1A promoter region, displayed both reduced steady-state levels of early gene mRNAs (E1A, E1B, E2A, E3, and E4) and decreased rate of viral replication in ST cells. Interestingly, mutant PAdV-3s containing the left-terminal 312 bp displayed increased transcription of early genes including E1A. Our results suggest that the left ITR of PAdV-3 contain the promoter like elements and the sequences (between nt 151 and 312) downstream of left ITR can enhance its promoter activity.

Antitumor effects of fusions composed of dendritic cells and fibroblasts transfected with genomic DNA from tumor cells.

Based on several previous studies indicating that transfection of genomic DNA can stably alter the character of the cells that take up the exogenous DNA, we investigated antitumor immunity conferred by fusions of syngeneic dendritic cells (DCs) and allogeneic fibroblasts (NIH3T3) transfected with genomic DNA from B16 tumor cells. Fusion cells (FCs) composed of dendritic and genetically engineered NIH3T3 cells were prepared with polyethylene glycol, and fusion efficiency was 30.3%. Prior immunization with FCs prevented tumor formation upon challenge with B16 tumor cells. Efficacy was reduced when studies were performed in mice depleted of NK cells. Vaccination with FCs containing DCs and fibroblasts transfected with denatured DNA did not inhibit tumor growth. Cytotoxic T cell (CTL) activity of spleen cells from immunized mice against both Yac-1 and tumor cells was also stimulated by administration of FCs compared with the activity observed for cells obtained from naïve mice. These data demonstrate the therapeutic efficacy of fusion cell-based vaccine therapy using syngeneic DCs and allogeneic fibroblasts transfected with tumor-derived genomic DNA.

Cancer therapy with DNA-based vaccines

The development of DNA-based vaccines arises from the knowledge that weakly immunogenic, tumor-associated antigens (TAAs), the products of mutant or dysregulated genes in the malignant cells, are expressed in a highly immunogenic form by antigen presenting cells. We successfully prepared vaccines that were effective in the treatment of cancer in mice by transfection of DNA from breast cancer cells into a mouse fibroblast cell line (LM). Fibroblasts express MHC class I-determinants along with B7.1, a co stimulatory molecule. (Classic studies indicate that transfection of genomic DNA can stably alter both the genotype and the phenotype of the cells that take-up the exogenous DNA.) The fibroblasts were transfected with sheared, unfractionated genomic DNA from a breast adenocarcinoma that arose spontaneously in a C3H/He mouse (H-2 k). To increase their non-specific immunogenic properties, the fibroblasts were modified before transfection to express allogeneic MHC-determinants (H-2K b) and to secrete IL-2. Afterward, the IL-2-secreting semi allogeneic cells were co transfected with DNA from the spontaneous breast neoplasm, along with a plasmid (pHyg) conferring resistance to hygromycin. Pooled colonies of hygromycin-resistant cells were then tested in C3H/He mice for their immunotherapeutic properties against the growth of the breast neoplasm. The results indicated that tumor-bearing mice immunized with the transfected cells survived significantly longer than mice in various control groups. Similar beneficial effects were seen in C57BL/6 mice injected with a syngeneic melanoma cells and semi allogeneic, IL-2-secreting fibroblasts transfected with DNA from the melanoma cells. The immunity was mediated by CD8 + T cells and was specific for the type to tumor from which the DNA was obtained.

A simple and rapid method for preparation of viral DNA from cell associated cytomegalovirus

In the field of human cytomegalovirus pathogenesis there is growing interest in analyzing recent clinical isolates rather than cell culture adapted laboratory strains. However, true low passage isolates are strictly cell associated prior to cell culture adaptation and only a minor fraction of cells are infected at low passage number. Both conditions hinder the preparation of pure viral DNA. To date, genetic analyses had been carried out mostly with supernatant associated cytomegalovirus. A rapid and simple method is described for preparation of viral DNA from low passage cell associated isolates with little cytopathogenic effect. The protocol is based on a combination of Triton X-100 lysis, nuclease treatment, and subsequent phenol chloroform extraction. Cellular background was reduced significantly to enable clear detection of all viral DNA fragments in restriction fragment length analysis. The method yielded DNA which was suitable for downstream applications like cloning of viral DNA fragments or transfection of genomic viral DNA. This method may facilitate genomic analyses of pathogenic cell associated recent cytomegalovirus isolates.

Treatment of Breast Cancer with Fibroblasts Transfected with DNA from Breast Cancer Cells

This investigation was based on the hypothesis that weakly immunogenic, breast cancer-associated Ags, the products of mutant or dysregulated genes in the malignant cells, will be expressed in a highly immunogenic form by semiallogeneic IL-2-secreting fibroblasts transfected with DNA from breast cancer cells. (Classic studies indicate that transfection of genomic DNA can stably alter both the genotype and the phenotype of the cells that take up the exogenous DNA.) To investigate this question, we transfected LM mouse fibroblasts (H-2k) modified to secrete IL-2 with genomic DNA from a breast adenocarcinoma that arose spontaneously in a C3H/He mouse (H-2k). To increase their nonspecific immunogenic properties, the fibroblasts were also modified before transfection to express allogeneic MHC determinants (H-2Kb). Afterward, the IL-2-secreting semiallogeneic cells were cotransfected with DNA from the spontaneous breast neoplasm, along with a plasmid (pHyg) conferring resistance to hygromycin. Pooled colonies of hygromycin-resistant cells were then tested in C3H/He mice for their immunotherapeutic properties against the growth of the breast neoplasm. The results indicated that tumor-bearing mice immunized with the transfected cells survived significantly longer than mice in various control groups. Similar beneficial effects were seen in C57BL/6 mice injected with a syngeneic breast carcinoma cell line (EO771) and semiallogeneic, IL-2-secreting fibroblasts transfected with DNA from EO771 cells. The immunity was mediated by CD8+ T cells since immunized mice depleted of CD8+ cells failed to resist tumor growth.

Mechanistic analyses of ion dependences in a high-affinity human serotonin transport system in transfected murine fibroblast cells.

1. A clonal cell line, L-S1, has been identified from transfection of human genomic DNA into cultured mouse L-M fibroblasts. Because this transfectant cell line stably expresses a high-affinity serotonin (5-HT) transport mechanism with kinetic and pharmacological properties comparable to those of other serotonin uptake systems, it was used to investigate the mechanistic involvement of Na+ and Cl- ions in the ligand binding and kinetic uptake processes of this system. 2. Intact transfectant cells, when incubated at low temperature (4 C), enabled quantitative assessment of imipramine-displaceable 5-[3H]HT binding to the 5-HT transport system. This binding activity is insensitive to the presence of various ligands specific for 5-HT receptor subtypes. 3. Imipramine-displaceable 5-[3H]HT binding to intact L-S1 cells was shown to be a Cl--dependent but Na+-independent process. Chloride ions lack binding co-operativity in facilitating ligand binding. Changes in external Cl- concentration altered the Kd but not the Bmax of binding. 4. The overall transport activity was observed to be highly dependent on both external Na+ and Cl- concentrations, characterized by a 5-HT:Na+:Cl- coupling ratio of 1:1:1 per transport cycle. Alterations in the external concentrations of both Na+ and Cl- ions altered only the Km and not the Vmax of transport. 5. Both binding and kinetic results are consistent with kinetic modelling predictions of the Cl- ion in facilitating 5-HT binding to the transport system, and of the Na+ ion in enabling translocation of bound 5-HT across the plasma membrane. Thus, Na+ and Cl- ions facilitate mechanistically distinct and discernible functions in the transport cycle.

Rescue of targeted regions of mammalian chromosomes by in vivo recombination in yeast.

In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in approximately 1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system.

A BALB/c 3T3-transformed cell line suitable for transfection assay of metastasis-inducing genes.

A clonal cell line, 1-1ras1000, transformed by the activated c-Ha-ras oncogene, does not form metastases after i.v. injection into mice (experimental metastasis assay). Here, we show that this cell line is useful as a recipient to detect metastasis-inducing genes, using a transfection assay. Cells (1-1ras1000) were susceptible to metastasis induction by transfection with either v-src or genomic DNA from a v-src-and v-fos-transferred highly metastatic rat cell line (SR202). The susceptibility of 1-1ras1000 cells for lung metastasis induction was suitable for a genomic transfection assay to detect a metastasis-inducing gene in the transfected cells which had incorporated genomic DNA from donor metastatic tumor cells. When DNAs extracted from 7 human tumors were tested for metastasis induction, 2 DNAs from nonmalignant tumors (non-tumorigenic tumors in athymic nude mice) (2/2) were negative and 4 DNAs from malignant tumors (4/5) were positive in 1-1ras1000 cells for primary transfection. in one of the resulting metastases, the ability to metastasize was also transferred in the second and third cycles of genomic DNA transfection at high frequencies. All of the resulting metastases carried the human repetitive Alu sequence. Neither re-arrangements of the endogenous c-Haras nor changes of protein amounts were detected. Recipient 1-1ras1000 cells had a negligible rate of spontaneously metastatic conversion during in vitro cultivation and transfection processes. The resulting metastasized cells were easily isolated from the lung after culturing in selection medium containing G418 (geneticin). Isolated cells stably retained the ability to form metastatic lung nodules when re-injected into mice. Thus, 1-1ras1000 cells appear to be a useful system for the isolation of metastasis-inducing genes from human metastatic tumors.

Evidence that the murine AIDS defective virus does not encode a superantigen

The T-cell receptor repertoire was analyzed in C57BL/6 mice upon infection with helper-free stocks of the pathogenic murine AIDS (MAIDS) defective virus in order to demonstrate if, as previously reported, this virus encodes a superantigen. A polyclonal T-cell stimulation involving T cells expressing multiple V beta subsets occurred within the first week of infection, while late in the disease we could note only a 50% deletion of V beta 5 CD8+ cells. Transfection of the MAIDS virus genomic DNA into fibroblasts and B cells expressing major histocompatibility complex class II molecules failed to show any stimulation of cells expressing the specific V beta (V beta 5) previously reported to respond to MAIDS virus-infected cells. In addition, mice lacking V beta 5 cells did not show any significant decrease in susceptibility to the disease compared with mice expressing V beta 5 and bred on the same genetic background. Our in vivo and in vitro results fail to demonstrate a role for a superantigen encoded by the MAIDS defective viral genome in the pathogenesis of MAIDS.