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PubMed2003

Characterization of a novel intracellular endopeptidase of the alpha/beta hydrolase family from Streptomyces coelicolor A3(2).

In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsin-like activity, absent from the wild type, was detected. Complementation that restored proteasome function did not suppress expression of the endopeptidase. Since the enzyme was not found in two other S. coelicolor proteasome mutants, its expression probably resulted from a secondary mutation arisen in the proteasome mutant. Purification of the endopeptidase revealed its identity to SCO7095, a putative hydrolase encoded by the S. coelicolor A3(2) genome with no known homologue. Based on the prediction of a Ser-Asp-His catalytic triad and an alpha/beta hydrolase fold, SCO7095 was assigned to peptidase clan SC. N-terminally His-tagged SCO7095 was efficiently expressed in Escherichia coli cells and purified for further characterization. Although SCO7095 is distantly related to several proline iminopeptidases, including Thermoplasma acidophilum tricorn-interacting F1, no aminopeptidase activity was detected. On synthetic substrates, the monomeric enzyme exhibited not only chymotrypsin-like activity but also thrombin-like activity.

Amino Acid Sequence↗

PubMed2000

Thermostable lipase of Bacillus Stearothermophilus: high-level production, purification, and calcium-dependent thermostability.

An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET-22b(+) expression vector and its expression was induced by IPTG at 30 degrees C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26% of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62%. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68 degrees C and it was stable up to 55 degrees C for 30 min-incubation. The thermostability increased by about 8-10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66 degrees C in the presence of calcium ions but at 58 degrees C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures.

Base Sequence↗

PubMed2005

Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K(D) approximately 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni(2+)-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M5), derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native antigen NcSRS2. We believe that the presented concepts offer convenient methods to achieve efficient adjuvant association of recombinant immunogens, and the advantages and disadvantages of the two concepts are discussed.

Animals↗

PubMed1991

Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.

Cloning, Molecular↗

PubMed1998

Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase.

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B-D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP-GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.

Amino Acid Sequence↗

PubMed1979

Mechanical efficiency of fast- and slow-twitch muscle fibers in man during cycling.

The influence of different percentages of slow-twitch (ST) and fast-twitch (FT) fibers in vastus lateralis on delta efficiency expressed by delta work (x)/delta energy liberation (y) in y = a + bx was studied in six subjects during cycling on an ergometer at 60 or 100 rpm at work loads below 80% of VO2max. Three subjects had an average of 78% ST fibers (ST group) and the other subjects had an average of 76% FT fibers (FT group). There was no difference between the two groups in delta efficiency at 60 rpm, but at 100 rpm the efficiency of the ST group was significantly lower than that of the FT group (19.6 vs. 28.8%, P less than 0.01). In the ST group respiratory exchange ratio (R) was higher at 100 rpm than at 60 rpm, but the FT group had similar R values at both pedal revolution rates. The most important finding was the reduced efficiency when pedaling frequency was increased from 60 to 100 rpm in the ST group (23.3 to 19.6%). Predominant use of ST fibers at rapid pedal rates may require a substantial increase in energy expenditure.

Adult↗

PubMed1995

Reporter gene expression upon stable transfection when only a TATA box or a TATA box plus Sp1 sites are present 5' to the gene.

Episomal plasmids for stable transfection of mammalian cell cultures were constructed that have a G418-resistance (neo) gene immediately downstream of a highly truncated promoter. These plasmids had a function hygromycin-resistance gene (hyg) as a selectable marker. Surprisingly, in LTK- cells, but not HeLa cells, stably transfected with these BK virus-based plasmids having no promoter elements adjacent to the neo gene, readthrough transcription, probably from about 1 kb upstream, gave almost as efficient expression of the neo gene as of the hyg gene with a full-length promoter immediately upstream. When the transfecting plasmids contained Epstein-Barr virus (EBV) DNA sequences for episomal maintenance and had multiple Sp1 sites and a TATA box as the only promoter elements 5' to the neo gene, only about 3-9% of HeLa transfectants were G418 resistant (G418R). In transfections with analogous plasmids lacking these promoter elements 5' to the neo gene, no G418R colonies were seen. The establishment of the G418R phenotype probably required integration of plasmid DNA into favorable chromosomal sites and was aided by the presence of the TATA box plus Sp1 sites as a subminimal promoter. The absence of detectable G418-resistance in most of the HeLa transfectant clones obtained with EBV-type plasmids, even at a high plasmid copy number and even when a TATA box and six Sp1 sites were present immediately upstream of the neo gene, indicates that these elements do not suffice for appreciable gene expression in vivo and that this is a suitable model system for studying DNA rearrangements that can potentiate expression of the neo gene.

Animals↗

PubMed1985

Expression of antibody-forming cell clones in two-stage cultures: an attempt to separate proliferation and maturation events.

Peripheral blood lymphocytes (PBL) from non-immunized rabbits or from rabbits immunized several months previously with a single dose of sheep red blood cells (SRBC) were cultured at several different cell densities in the presence of antigen for 5 days. The cells were then distributed in 10 microliter microcultures at different cell densities to estimate the frequency of responding units. It was found that a shift up in cell concentration from first to second stage allows a more efficient expression of antibody-forming cell clones. We conclude that, during the first stage at low cell density, the precursor cells proliferate and, when they are partitioned in microcultures and the cell concentration is raised, maturation to antibody-forming cells occurs. This approach allows a separation of the proliferation event from the maturation event and it may prove to be a useful tool in the study of B-cell differentiation.

Animals↗

PubMed2005

Cloning and expression of Klebsiella phage K11 lysozyme gene.

Previously, the lysozyme gene of the Klebsiella phage K11 was partially sequenced in our lab. Using the sequence information the lysozyme gene of the Klebsiella phage K11 was amplified and cloned using the polymerase chain reaction of the pfu DNA polymerase. The nucleotide sequence of phage K11 lysozyme gene was determined. The open reading frame corresponds to a polypeptide with 151 amino acids and molecular weight of 16,932 Da. The deduced amino acid sequence of this polypeptide shows 74-75% homologies to the T7 and T3 phage lysozymes. Although the gene was efficiently expressed under the control of tac promoter in Escherichia coli XL1-blue cells at 37 degrees C, most of the K11 lysozyme produced was insoluble. When the temperature of cell growth was lowered, however, solubility of the K11 lysozyme was increased gradually. The insoluble protein expressed at 37 degrees C was solubilized in 5 M guanidine-HCl and refolded in the presence of oxido-shuffling agent (GSH/GSSG). Through the refolding process the recombinant lysozyme was solubilized and purified. The purified K11 lysozyme showed transcription inhibition of K11 RNA polymerase as well as amidase activity. These results showed that the lysozyme of bacteriophage K11 is a bifunctional protein that cuts a bond in the bacterial cell wall and selectively inhibits K11 phage RNA polymerase. Also, transcription inhibition ability of K11 lysozyme with T7 or SP6 phage RNA polymerase was measured. T7 RNA polymerase was less inhibited than K11 RNA polymerase by K11 lysozyme. But SP6 RNA polymerase was not nearly inhibited by K11 lysozyme.

Amidohydrolases↗

PubMed2005

Hemagglutinating virus of Japan-artificial viral envelope liposome-mediated cotransfer of bag-1 and bcl-2 genes protects hepatic cells against ischemic injury through BAG-1-assisted preferential enhancement of bcl-2 protein expression.

Hepatic injury subsequent to ischemia-reperfusion (I/R) was demonstrated in our previous study to be prevented by hemagglutinating virus of Japan (HVJ)-artificial viral envelope (AVE) liposome-mediated gene transfer of the antiapoptotic gene, human bcl-2 (h-bcl-2). In the present study, we introduced simultaneously both mouse Bcl-2-associated athanogene 1 (m-bag-1) and the h-bcl-2 gene by the same HVJ-AVE liposome transfection method, and found that I/R-induced hepatic injuries such as release of hepatic marker enzymes into blood, cell morphological degeneration, and cellular DNA strand cleavage were suppressed more effectively than by transfection with either gene singly. In addition, the h-Bcl-2 expression level in the ischemic state, but not in the nonischemic state, was markedly higher in h-bcl-2/m-bag-1-cotransfected liver than in h-bcl-2-transfected liver. In contrast, the m-BAG-1 expression level in the ischemic state, but not in the nonischemic state, was only slightly higher in h-bcl-2/m-bag-1-cotransfected liver than in m-bag-1-transfected liver. Thus, with dual gene cotransfer, coexistent Bcl-2 protein exerts no activity to assist a marked enhancement of BAG-1 protein, whereas the function of overexpressed BAG-1 as a Bcl-2-binding protein may lead to the enhancement of efficient expression of h-Bcl-2 in I/R-treated liver as compared with nonischemic liver, which results in repression of diverse I/R-induced cell death symptoms, presumably through the formation of functional complexes of BAG-1 and Bcl-2.

Animals↗

PubMed2004

Role for CCR5Delta32 protein in resistance to R5, R5X4, and X4 human immunodeficiency virus type 1 in primary CD4+ cells.

CCR5Delta32 is a loss-of-function mutation that abolishes cell surface expression of the human immunodeficiency virus (HIV) coreceptor CCR5 and provides genetic resistance to HIV infection and disease progression. Since CXCR4 and other HIV coreceptors also exist, we hypothesized that CCR5Delta32-mediated resistance may be due not only to the loss of CCR5 function but also to a gain-of-function mechanism, specifically the active inhibition of alternative coreceptors by the mutant CCR5Delta32 protein. Here we demonstrate that efficient expression of the CCR5Delta32 protein in primary CD4(+) cells by use of a recombinant adenovirus (Ad5/Delta32) was able to down-regulate surface expression of both wild-type CCR5 and CXCR4 and to confer broad resistance to R5, R5X4, and X4 HIV type 1 (HIV-1). This may be important clinically, since we found that CD4(+) cells purified from peripheral blood mononuclear cells of individuals who were homozygous for CCR5Delta32, which expressed the mutant protein endogenously, consistently expressed lower levels of CXCR4 and showed less susceptibility to X4 HIV-1 isolates than cells from individuals lacking the mutation. Moreover, CD4(+) cells from individuals who were homozygous for CCR5Delta32 expressed the mutant protein in five of five HIV-exposed, uninfected donors tested but not in either of two HIV-infected donors tested. The mechanism of inhibition may involve direct scavenging, since we were able to observe a direct interaction of CCR5 and CXCR4 with CCR5Delta32, both by genetic criteria using the yeast two-hybrid system and by biochemical criteria using the coimmunoprecipitation of heterodimers. Thus, these results suggest that at least two distinct mechanisms may account for genetic resistance to HIV conferred by CCR5Delta32: the loss of wild-type CCR5 surface expression and the generation of CCR5Delta32 protein, which functions as a scavenger of both CCR5 and CXCR4.

Adenoviridae↗

PubMed1982

Metabolic and mechanical effects using L- and D-carnitine in working swine hearts.

The purpose of the present studies was to contrast the differences in mechanical and metabolic functions between treatments of L- and D-carnitine as measured in intact, regionally ischemic swine hearts. Coronary flows were decreased in the anterior descending circulation (-53% delta) after an equilibration period of 30 min during which hearts in both treatment groups were supplemented with trace-labeled [U14-C]palmitate. Nine hearts were selectively treated with L-carnitine (avg serum concentration 804 nmol/ml) and were compared with eight hearts treated with D-carnitine (same dosage schedule). No differences in fatty acid metabolism or regional shortening were observed between treatment groups in the heart muscle rendered moderate-to-severely ischemic. L-Carnitine did effect a significant reduction in fatty acid uptake and labeled CO2 production as compared with D-carnitine in aerobic tissue. This resulted in a secondary improvement in global mechanical efficiency (expressed as a ratio of either left ventricular developed pressure or work/global oxygen consumption) in L-treated hearts. Higher levels of high-energy phosphate stores were also noted in both ischemic and aerobic tissue of L-treated hearts. Thus differences in fatty acid metabolism occurred in working swine hearts based on the structural specificity of carnitine treatment. L-Carnitine favorably modified oxygen consumption and global mechanical efficiency.

Animals↗

PubMed1991

Overexpression of the human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors.

The human vitamin D3 receptor (hVDR) cDNA was cloned into the E1 region of the adenovirus genome to generate recombinant viruses which were used to infect 293 (adenovirus-transformed human fetal kidney) cells. High salt extracts from cells infected with the recombinant viruses were subjected to immunoblot analysis using a monoclonal antibody to chicken VDR and were shown to contain large quantities of a protein of approximately 50 kDa with a migration identical to that of the hVDR in T47D (human mammary adenocarcinoma) cells. Scatchard analysis showed that the infected cells express approximately 100-fold more receptor than T47D cells and that this receptor binds 1,25-dihydroxyvitamin D3 with high affinity. The overexpressed hVDR also binds to DNA-cellulose and is eluted with a KCl concentration similar to that determined for fully active endogenous VDR. Nuclear extracts from cells infected with the hVDR-expressing adenoviruses contain an activity that specifically binds an oligonucleotide with sequences from the rat osteocalcin vitamin D3 response element, as determined by gel mobility shift. This interaction can be inhibited by the presence of an anti-VDR antibody, but not by nonspecific immunoglobulins. We conclude, therefore, that the overexpressed receptor has the ligand- and DNA-binding characteristics defined for endogenous VDR and that adenoviruses can be used to efficiently express large quantities of functional hVDR in a human cell line. Finally, a second binding activity, specific for the vitamin D response element, but distinct from the VDR, has been identified in extracts from uninfected cells.

Adenoviruses, Human↗

PubMed2003

[Construction of eukaryotic expression vector of hMTH1 gene antisense RNA].

OBJECTIVE: To construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA. METHODS: The conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast (HLF) and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection. RESULTS: 423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology. Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%. CONCLUSION: The efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.

DNA Repair Enzymes↗

PubMed1990

An intragenic region downstream from the dihydrofolate reductase promoter is required for replication-dependent expression.

The gene encoding dihydrofolate reductase (DHFR) is down-regulated as myoblasts withdraw from the cell cycle and commit to terminal differentiation. To localize cis-acting elements involved in regulating DHFR gene expression, the DHFR promoter and upstream region, together with differing amounts of contiguous intragenic sequence, were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The resulting fusion genes were stably transformed into muscle cells, and CAT mRNA levels were measured in proliferative myoblasts and committed myocytes. A gene consisting of the -850/+465 region of DHFR (numbers refer to distance in base pairs from transcription initiation site) fused to CAT was efficiently expressed in proliferating myoblasts and was appropriately down-regulated during commitment. A gene consisting of the -850/+60 region of DHFR fused to CAT was poorly expressed in proliferating myoblasts and was not down-regulated during commitment. When inserted between the Rous sarcoma virus promoter and CAT sequence of RSVpCAT, the +61/+465 region of the DHFR gene augmented CAT mRNA expression in muscle cell transformants but did not confer a regulated pattern of expression. Our data indicate that DHFR sequences between +60 and +465 are required but are not sufficient for replication-dependent expression. The DHFR sequences may be operating at either a transcriptional or posttranscriptional level.

Animals↗

PubMed1991

In vitro expression of a chimeric coat protein gene from Grapevine Fanleaf virus (strain F 13).

The coat protein (CP) cistron of Grapevine Fanleaf virus strain F13 (GFLV-F13) has been located in the C-terminal region of the 122k polyprotein encoded by the genomic RNA 2 [Serghini et al. (1990) J. Gen. Virol. 71: 1433-1441]. A chimeric CP gene of GFLV-F13 including a short sequence corresponding to 3 restriction sites, the leader sequence of the GFLV-F13 satellite RNA and an initiation codon was constructed. Transcripts from this construct were translated in wheat germ extract with equal efficiency to form a 56k protein which comigrates on PAGE with the GFLV-F13 CP and a protein of 52k. Both species react with GFLV-F13 CP-specific antibodies. Deletions in the 5' region of the CP gene show that the 56k protein is initiated at the first AUG after the satellite leader and the 52k protein at the second in-frame AUG. Transcripts with a 142 nt deletion including the two AUG codons from the 5' end of the CP gene are not efficiently expressed in vitro, no major translation product being detected.

Base Sequence↗

PubMed2004

Antigen epitope of Helicobacter pylori vacuolating cytotoxin A.

AIM: To construct and select antigen epitopes of vacuolating cytotoxin A (VacA) for nontoxic VacA vaccine against Helicobacter pylori (H pylori ) infection. METHODS: Eleven VacA epitopes were predicted according to VacA antigenic bioinformatics. Three candidates of VacA epitope were constructed through different combined epitopes. The candidate was linked with E. coli heat-labile enterotoxin B (LTB) by a linker of 7 amino acids, and cloned into plasmid pQE-60 in which fusion LTB-VacA epitope was efficiently expressed. To test the antigencity of the candidate, 6 BALB/c mice were treated with the fusion LTB-VacA epitope through intraperitoneal injection. To explore the ability of inhibiting the toxicity of VacA,cantiserum against the candidate was used to counteract VacA that induced HeLa cells to produce cell vacuoles in vitro. RESULTS: Serum IgG against the candidate was induced in the BALB/c mice. In vitro, the three antisera against the candidate efficiently counteracted the toxicity of VacA, and decreased the number of cell vacuoles by 14.17%, 20.20% and 30.41% respectively. CONCLUSION: Two of the three candidates, LZ-VacA1and LZ-VacA2, can be used to further study the mechanism of vacuolating toxicity of VacA, and to construct nontoxic VacA vaccine against H pylori infection.

Amino Acid Sequence↗

PubMed1992

A spleen necrosis virus-based retroviral vector which expresses two genes from a dicistronic mRNA.

We have investigated a novel strategy for coexpressing two genes from a retroviral vector. The 5' nontranslated leader region of at least some picornavirus RNAs contains a sequence that can act as an internal ribosome entry site allowing initiation of translation at a downstream AUG codon in a 5' cap-independent manner. To investigate whether such a sequence can function in the context of a retroviral vector, we constructed a spleen necrosis virus-based vector carrying two selectable marker genes separated by the leader region of encephalomyocarditis virus. This vector was genetically stable and efficiently expressed both markers from a single dicistronic transcript. Since the expression of two genes by other strategies in retroviral vectors can often be problematic, these results offer a promising new approach for the design of "double gene" retroviral vectors.

Animals↗