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At least 145 records · Page 8Linked to original sources
PubMed1992

Identification of a generalised packaging sequence for D-type retroviruses and generation of a D-type retroviral vector.

In order to construct vectors based upon D-type, rather than C-type, retroviruses, we have identified a 624-bp fragment of Mason-Pfizer monkey virus (MPMV) which constitutes a packaging sequence for at least two D-type retroviruses. When this fragment was included in an extensively deleted D-type vector genome, the D-type viruses MPMV and SRV-5, but not the C-type viruses MLV-A or MLV-E, rescued the vector RNA from HeLa cells. The recombinant virus stocks have the host range of the rescuing D-type virus as shown by expression of an internal (SV40-puromycin) cassette replacing the retroviral structural genes. The recombinant MPMV was specifically neutralized by anti-MPMV serum and receptor interference was demonstrated when it was plated on cells productively infected with wild type MPMV. When the putative D-type packaging sequence was removed from the vector genome, even though the other sequence elements required for efficient reverse transcription remained, the vector was no longer rescued from HeLa cells. These results complement the recent demonstration of broad specificity of rescue of a C-type vector (carrying only the packaging sequence of Mo-MLV) by several different C-type, but not D-type, viruses. Replacement of the D-type packaging sequence by most of the extended packaging sequence of Mo-MLV prevented the otherwise D-type vector from being rescued by D-type viruses and did not allow it to be rescued by C-type viruses. This was probably because of the incompatibility of the D-type vector sequences with the C-type retroviral proteins involved in viral reverse transcription and integration. Hence, we have localized a packaging sequence that is recognized by D-type, but not by C-type, retroviruses and have constructed a D-type vector which may be useful in gene transfer experiments.

Betaretrovirus↗
PubMed1991

A system, using neural cell lines, to characterize HSV-1 vectors containing genes which affect neuronal physiology, or neuronal promoters.

Among the potential uses of defective herpes simplex virus (HSV-1) vectors are to study neuronal physiology, neuronal gene regulation, and to perform gene therapy of neuronal diseases. The prototype HSV-1 vector, pHSVlac, stably expresses Escherichia coli beta-galactosidase from the HSV-1 immediate early (IE) 4/5 promoter in cultured rat peripheral and CNS neurons, and in neurons in the adult rat brain. The LacZ gene and the IE 4/5 promoter in pHSVlac can be replaced with genes which affect neuronal physiology or cellular promoters, respectively. A system is required to characterize these HSV-1 vectors; cultured neurons, a mixture of different kinds of neurons and glia, cannot be used. In contrast, neural cell lines represent a homogenous population of neural cells available in virtually unlimited quantities. A system, using neural cell lines, to characterize HSV-1 vectors carrying other genes or promoters is now reported: First, 4 assays are described to detect HSV-1 vector DNA, RNA transcribed from the vector, and to quantitate beta-galactosidase expression. Second, 8 cell lines derived from rodents, primates, and humans were infected with pHSVlac virus and shown to express beta-galactosidase. The cell lines tested included adrenergic and cholinergic mouse neuroblastoma cells, rat pheochromocytoma cells, rodent pituicytes, and human neuroblastoma cells. Infection of these cell lines should prove useful for characterizing HSV-1 vectors with molecular and biochemical assays. Third, differentiated rat pheochromocytoma and mouse neuroblastoma cells, which resemble neurons, were infected with pHSVlac virus and shown to stably express beta-galactosidase. Infection of these cells should be useful for determining the effect of various HSV-1 vectors on neuronal physiology. Thus, HSV-1 vectors containing various genes or promoters can be characterized using the system described in this study.

Animals↗
PubMedDate not supplied

The use of integrating DNA vectors to analyse the molecular defects in ionising radiation-sensitive mutants of mammalian cells including ataxia telangiectasia.

Integrating DNA vectors, encoding selectable recombinant genes, were used to assess rejoining and recombination in wild-type mammalian cells and their ionising radiation-sensitive mutants. To provide a simple model of an important radiation-induced lesion - the DNA double-strand break - the vectors were cut with restriction endonucleases at specific single sites. If these breaks were made in the coding sequence of a selectable gene, the fidelity of the rejoin/recombination process could be measured by survival of vector-transformed cells in selective medium. Rejoining was assessed using vectors without internal homologies, while recombination was measured using pairs of fragments or deletion vectors carrying homologous regions. Initial experiments were made with vectors carrying a single selectable gene but, to overcome potential artefacts, 2-gene vectors were then constructed where one gene acts as a linked marker and (unbroken) control for the other (broken) gene. Available data are reviewed to show that, compared to their respective wild-type counterparts: (1) an ataxia telangiectasia (A-T) cell line and the hamster irs1 mutant show a consistent reduction in the fidelity of rejoining double-strand breaks (while the hamster mutants irs2, irs3, xrs series, and EM9 show wild-type fidelity); (2) the hamster EM9 mutant shows a reduction in ability to recombine homologous vector fragments (while the A-T line and probably the xrs mutants show show wild-type abilities); and (3) the xrs mutants show a reduction in overall transformation frequency with vector DNA, whether broken or not, while the other mutants tested show approximately wild-type frequencies. A critical account of the techniques and data is given, together with speculations on the molecular nature of the processes which are defective in these mutants, leading to radiosensitivity.

Ataxia Telangiectasia↗
PubMed1988

Bunyavirus-vector interactions.

Recent advances in the genetics and molecular biology of bunyaviruses have been applied to understanding bunyavirus-vector interactions. Such approaches have revealed which virus gene and gene products are important in establishing infections in vectors and in transmission of viruses. However, much more information is required to understand the molecular mechanisms of persistent infections of vectors which are lifelong but apparently exert no untoward effect. In fact, it seems remarkable that LAC viral antigen can be detected in almost every cell in an ovarian follicle, yet no untoward effect on fecundity and no teratology is seen. Similarly the lifelong infection of the vector would seem to provide ample opportunity for bunyavirus evolution by genetic drift and, under the appropriate circumstances, by segment reassortment. The potential for bunyavirus evolution by segment reassortment in vectors certainly exists. For example the Group C viruses in a small forest in Brazil seem to constitute a gene pool, with the 6 viruses related alternately by HI/NT and CF reactions, which assay respectively M RNA and S RNA gene products (Casals and Whitman, 1960; Shope and Causey, 1962). Direct evidence for naturally occurring reassortant bunyaviruses has also been obtained. Oligonucleotide fingerprint analyses of field isolates of LAC virus and members of the Patois serogroup of bunyaviruses have demonstrated that reassortment does occur in nature (El Said et al., 1979; Klimas et al., 1981; Ushijima et al., 1981). Determination of the genotypic frequencies of viruses selected by the biological interactions of viruses and vectors after dual infection and segment reassortment is an important issue. Should a virus result that efficiently interacts with alternate vector species, the virus could be expressed in different circumstances with serious epidemiologic consequences. Dual infection of vectors with different viruses is not unlikely, because many bunyaviruses are sympatric in nature. For example, the Ae. trivittatus-cottontail rabbit and the Ae. triseriatus-squirrel arbovirus cycles are sympatric in the ecotone between their respective grassland and forest ecosystems (LeDuc, 1979). Should a LaCrosse virus variant or reassortant evolve that was efficiently vectored by Ae. trivittatus mosquitoes, significantly more human infections with La Crosse virus would likely occur. Unlike Ae. triseriatus, Ae. trivittatus mosquitoes are not restricted to forested areas and consequently are more likely to encounter and to feed upon humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Aedes↗
PubMed1988

A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers.

Several families of positive-selection cloning vectors were constructed, based on the principle of palindrome nonviability first used by Hagan and Warren [Gene 19 (1982) 147-151]. Each vector, derived from either pBR322 or RSF1010 (a broad-host-range plasmid), contains a long inverted repeat (2 x 366 to 2 x 1008 bp) ending in a symmetrical polylinker. Plasmids with long palindromes are not viable in most strains of Escherichia coli and in at least one Gram-positive bacterium. These palindrome-containing vectors therefore transform such strains at a very low frequency unless a DNA fragment is cloned within the polylinker at the center of the palindrome. Transformation by plasmids lacking an insert is reduced by two to four orders of magnitude. Such vectors can be propagated in a palindrome-tolerant strain; however, long symmetrical deletions then occur within the palindrome. To suppress the resulting deletion derivatives, vectors have been constructed so that an extensive deletion would remove the selectable marker. Alternatively, the vectors can be propagated in any strain of E. coli so long as the palindrome is interrupted by a nonpalindromic DNA fragment. We also present several symmetrical polylinkers and drug-resistance cassettes within the vectors. These components can be interchanged to make new positive-selection vectors as needed, and the cassettes are useful in insertional mutagenesis as well. A general method is described to convert virtually any small or medium-sized plasmid into a positive-selection vector.

Base Sequence↗
PubMed1989

Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells.

A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.

Antigens, Viral↗
PubMed1991

A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing.

A new family of vectors including cloning vectors (pEK0; pEC5), an expression vector (pEKEx1), and promoter probe vectors (pEKpllacZ; pEKplCm), has been constructed. All these shuttle vectors are based on the replication origins of the corynebacterial pBL1 and the Escherichia coli ColE1 plasmids, and thus are able to replicate in Corynebacterium glutamicum and E. coli. Plasmids pEK0 and pEC5 carry multiple restriction sites useful for gene cloning and the kanamycin- or chloramphenicol-resistance-encoding gene from Tn903 or from Tn9, respectively. In C. glutamicum, both vectors are compatible with vectors containing the corynebacterial pHM1519 replicon. Based on plasmid pEK0, the expression vector pEKEx1 was developed to allow for isopropyl-beta-D-thiogalactopyranoside-inducible expression of inserted genes in C. glutamicum and E. coli. Also based on pEK0, the promoter probe vectors pEKpllacZ and pEKplCm were constructed to carry the promoterless lacZ or cat reporter genes downstream from useful cloning sites, for assaying the transcriptional activity of cloned fragments.

Cloning, Molecular↗
PubMed1995

Integrative vectors for heterologous gene expression in Streptomyces spp.

Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed. The vectors are comprised of a strong constitutive promoter, PE, a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene. The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site. HyR transformants are stable in the absence of drug selection. Conjugative derivatives were also constructed by incorporating oriT, the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E. coli donor to Steptomyces lividans (Sl). Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described. Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems. The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl.

Base Sequence↗
PubMed1989

Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.

This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested, HUT 78 and Raji. These results are consistent with previous observations that upstream promoters exert an inhibitory effect on promoters placed downstream and bear out the predictions used in the design of DC vectors. The use of DC vectors may contribute to the solution of the problems encountered in expressing retrovirally transduced genes in cultured cells and, in particular, when introduced into the live animal.

Adenosine Deaminase↗
PubMed1992

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

Animals↗
PubMed1995

Pharmacokinetics of adenoviral vector-mediated gene delivery to vascular smooth muscle cells: modulation by poloxamer 407 and implications for cardiovascular gene therapy.

Regional in vivo delivery of therapeutic genes to the cardiovascular system at sites of localized vascular disease is feasible by catheter-mediated delivery of recombinant adenoviral vectors. Vascular smooth muscle cell (SMC) proliferation, which follows angioplasty and contributes to restenosis, is one process that may be amenable to such a gene therapy strategy. The clinical utility of localized delivery strategies such as this critically depends upon successful gene transfer to sufficient numbers of vascular cells, locally, within a clinically acceptable time period. Relatively limited information is available concerning the kinetics of gene transfer by first-generation, replication-deficient, recombinant adenovirus (Av1) vectors. In this context, we evaluated the pharmacokinetics of adenoviral vector-mediated gene delivery to vascular SMC using an Av1 reporter vector (Av1LacZ4) expressing a nuclear-targeted beta-galactosidase (beta-Gal) reporter. Bovine aortic SMC were exposed to Av1LacZ4 for various times at a range of concentrations and multiplicities of infection (MOI). After exposure, cells were washed and evaluated for transduction at 48 hr by X-Gal staining. Transduction occurred with a rate constant typically determined in the range of 10(-10) to 10(-11) events.ml/cell.virion.min. The rate of transduction was directly dependent on virion concentration, but not substantially on the virion-to-cell ratio. Relatively low fractions of the total input vector were found to be consumed, even after prolonged adsorption times. We hypothesized that the cellular transduction rate (and thus overall efficiency) would be improved by agents that could maintain a prolonged, high pericellular vector concentration. To evaluate this, cells were exposed to the vector in the presence of 15 grams/dl poloxamer 407, a viscous biocompatibile polyol, for various times followed by washout and evaluation as described above. Both cells and vector remained viable under these conditions, and poloxamer was found to increase the apparent transduction rate 10-fold or more (1-5 x 10(-9) transduction events.ml/cell.virion.min), with remarkable increases in numbers of cells transduced even after brief exposure periods. These observations demonstrate that the pharmacokinetics of adenoviral-mediated gene delivery to vascular SMC can be modulated by agents such as poloxamer 407, which may improve gene delivery by maintaining high pericellular concentrations of vector. Such modulation may permit achievement of desired levels of gene transfer while requiring lower total viral dosage and exposure time, and in turn may have important implications for in vivo gene delivery to vascular tissues.

Adenoviridae↗
PubMed1994

Improved self-inactivating retroviral vectors derived from spleen necrosis virus.

Self-inactivating (SIN) retroviral vectors contain a deletion spanning most of the right long terminal repeat's (LTR's) U3 region. Reverse transcription copies this deletion to both LTRs. As a result, there is no transcription from the 5' LTR, preventing further replication. Many previously developed SIN vectors, however, had reduced titers or were genetically unstable. Earlier, we reported that certain SIN vectors derived from spleen necrosis virus (SNV) experienced reconstitution of the U3-deleted LTR at high frequencies. This reconstitution occurred on the DNA level and appeared to be dependent on defined vector sequences. To study this phenomenon in more detail, we developed an almost completely U3-free retroviral vector. The promoter and enhancer of the left LTR were replaced with those of the cytomegalovirus immediate-early genes. This promoter swap did not impair the level of transcription or alter its start site. Our data indicate that SNV contains a strong initiator which resembles that of human immunodeficiency virus. We show that the vectors replicate with efficiencies similar to those of vectors possessing two wild-type LTRs. U3-deleted vectors carrying the hygromycin B phosphotransferase gene did not observably undergo LTR reconstitution, even when replicated in helper cells containing SNV-LTR sequences. However, vectors carrying the neomycin resistance gene did undergo LTR reconstitution with the use of homologous helper cell LTR sequences as template. This supports our earlier finding that sequences within the neomycin resistance gene can trigger recombination.

Base Sequence↗
PubMed1989

A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation.

A group of efficient cDNA cloning strategies employs vector-primers where cDNA synthesis starts from the oligo(dT)-primer tail, which is conventionally attached to cloning vectors by use of terminal deoxynucleotidyl transferase. An alternative, efficient and more versatile method of vector-primer preparation is to directly ligate, by use of T4 DNA ligase, a double-digested vector, e.g., pTZ18R/Pst I/Bam HI, to a synthetic (Bam HI)-adapter-end-primer, 5'-pGATCC-Tn or 5'-pGATCC-site-specific sequence. The use of a utility-vector containing a sizable spacer between the two selected restriction sites enables unambiguous separation on agarose gels of the double-digested vector precursors from single-digested ones, further simplifying the vector preparation. The adapter-end-primer ligation method can be applied to any suitable vectors with multiple cloning sites for the preparation of not only oligo(dT)-tailed, but also site-specific sequence-tailed vectors. Thus, the method enables the cDNA cloning of total poly (A+)-mRNAs, as well as specific RNA or mRNA species with or without poly(A)-tail.

Biotechnology↗
PubMedDate not supplied

[Vectors--derivatives of phage lambda for construction and analysis of genome libraries].

Basic features of lambda phage derived, cosmid and plasmid vectors are described. Plasmid vectors combine the most useful features of phage and plasmid vectors. Plasmids can exist in vivo as a plasmid or as a phage. Plasmid vectors are similar to large capacity phage vectors, but can be maintained in vivo without a stuffer fragment. That is why plasmids are easier in preparation for cloning than phage vectors. The yield of recombinants is higher with plasmid vectors (up to 3.10(6)) and the background of non-recombinants in the library is lower. Analysis of recombinant plasmids is more simple and effective than analysis of recombinant phages and plasmids. Probably plasmid vectors will soon be widely used instead of phage or cosmid vectors for genomic libraries construction and analysis.

Bacteriophage lambda↗
PubMed1995

Enhanced transduction efficiency of retroviral vectors coprecipitated with calcium phosphate.

Retroviral vectors are being used increasingly in clinical gene therapy protocols but low transduction frequencies are presenting a significant obstacle to progress. In this paper we report a simple method to enhance the efficiency of ex vivo retroviral gene transfer. Calcium chloride is added to the vector stock and calcium phosphate precipitates out of solution in complex with the retroviral vectors. When such coprecipitated vectors were used for gene transfer, the vector titres were increased at least five-fold and as much as 50-fold compared with the titres obtained in standard polybrene-enhanced infection protocols. Titre enhancement was not dependent on the starting concentration of vector, was equally effective for ecotropic and amphotropic vectors on a variety of mouse and human cells, and was not associated with any alteration in vector host range properties. The method may be of value to concentrate retroviral vectors and to enhance the efficiency of gene transfer in selected human gene therapy protocols.

Animals↗
PubMed1995

Foreign gene expression by human adenovirus type 5-based vectors studied using firefly luciferase and bacterial beta-galactosidase genes as reporters.

Adenovirus (Ad) vectors have been used extensively to obtain high-level expression of foreign genes in mammalian cells and are currently being studied for use as live viral-vectored vaccines and as gene transfer vectors for gene therapy. Many Ad recombinants have been generated that express foreign genes inserted in early region 3 (E3); however, little has been done to study the importance for gene expression of regulatory sequences flanking the gene. We have generated a series of Ad5 helper-independent vectors that contain the firefly luciferase gene or the bacterial beta-galactosidase gene (LacZ) with or without simian virus 40 (SV40) regulatory sequences, combined with E3 deletions of 1.88 or 2.69 kb. The greatest levels of luciferase expression were obtained with a vector containing the luciferase gene under the control of the SV40 promoter and polyadenylation signal inserted in a 1.88-kb E3 deletion. In contrast, LacZ expression was highest with a vector containing the LacZ gene with just the SV40 polyadenylation sequence combined with a 1.88-kb E3 deletion. It was also observed that regardless of the SV40 sequences flanking the reporter gene or the E3 deletion used, expression from the luciferase recombinants was dependent on viral DNA replication, whereas expression from the LacZ recombinants was only partially reduced when DNA replication was blocked. Analyses of RNA by dot blot hybridizations revealed that the levels of reporter gene-specific mRNA for various vectors in each series did not vary significantly. These results indicate that the kinetics and efficiency of expression of genes inserted into the E3 region, in nonconditional helper-independent vectors, may be more strongly dependent on the sequences in the foreign gene insert itself than on flanking regulatory sequences such as those used here, derived from SV40.

Adenoviruses, Human↗
PubMed1991

Effects of vector cutting on its recombination with the chromosomal immunoglobulin gene in hybridoma cells.

We have analyzed the effects of linearizing vector DNA on the frequency and pathway of its recombination with the homologous chromosomal gene. The pSV2neo vector bearing a 4.3-kb fragment encoding the mouse immunoglobulin mu heavy chain constant (C mu) region was cut either at sites within the C mu segment or outside C mu and then transferred to hybridoma cells bearing a mutant mu gene. The frequency of recombinant cells producing normal mu was then measured. For most cut sites, whether in regions of homology or of nonhomology, linearization of the transferred DNA enhanced the recombination frequency between the vector and chromosomal mu genes. When the vector was either uncut or cut at SacI in the region of homology, G418-resistant mu m+ recombinants were found to have integrated the vector by a single reciprocal homologous crossover; the enzyme site (SacI) used for cutting was present in the recombinants. By contrast, when the vector had been linearized at PvuI or SfiI in the region of nonhomology, vector integration involved nonhomologous crossovers, either between transferred DNA molecules or between transferred and chromosomal DNA, and the vector cut sites were absent in these recombinants. Some recombinants were found to have an unaltered as well as recombinant mu gene, suggesting that the nonhomologous recombination process might have involved sister chromatids.

Animals↗
PubMed1992

Improved gene expression by a U3-based retroviral vector.

To improve the expression of the genes transduced by retroviral vectors, we have constructed a U3-based retroviral vector and evaluated its effect on the expression of an insert from the internal promoter. The unique feature of the vector is that the transduced gene is inserted at the U3 region of the 3' long terminal repeats (LTR). Consequently, in the infected cells the gene is duplicated and transferred to the 5'-LTR. When compared with the conventional retroviral vectors which insert the gene within the retroviral transcriptional unit, the U3-based vectors greatly enhanced the expression of the transduced gene under all three promoters tested, viz. the cytomegalovirus immediately early gene promoter (CMV), the SV40 early gene promoter (SV), and the herpes simplex virus thymidine kinase gene promoter (TK). The SV and TK promoters which were previously shown suppressed by the retroviral promoter in the conventional construction restored their potencies in the U3-based vectors. Our results therefore suggested that the U3-based vectors are more advantageous than the conventional vectors for gene expression.

Animals↗