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At least 37 records · Page 2Linked to original sources
PubMed2003

Human dendritic cells genetically engineered to express cytosolically retained fragment of prostate-specific membrane antigen prime cytotoxic T-cell responses to multiple epitopes.

The ability of two plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses is demonstrated. The first vaccine (truncated; tPSMA) encodes for only the extracellular domain of prostate-specific membrane antigen (PSMA). The product, expressed following transfection with this vector, is retained in the cytosol and degraded by the proteasomes. For the "secreted" (sPMSA) vaccine, a signal peptide sequence is added to the expression cassette and the expressed protein is glycosylated and directed to the secretory pathway. Monocyte-derived dendritic cells (DCs) are transiently transfected with either sPSMA or tPSMA plasmids. The DCs are then used to activate autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs or with peptide-pulsed monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tPSMA DCs and sPSMA DCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted toward one of the four PSMA-derived epitopes when priming and boosting is performed with sPSMA. In contrast, tPSMA-transfected DCs prime T cells toward several PSMA-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance.

Antigens, Surface↗
PubMed2004

Viral interleukin-10-engineered autologous hematopoietic stem cell therapy: a novel gene therapy approach to prevent graft rejection.

The Epstein-Barr virus-encoded protein BCRF1 (viral interleukin [vIL]-10) is a biologically active homologue of cellular interleukin (IL)-10. In this study, a novel gene therapy approach to prolong allograft survival was designed. Autologous (syngeneic) hematopoietic progenitor/stem cell-enriched (HSC; lineage(-ve)) population derived from CBA/J mouse bone marrow were transduced with retrovirus encoding vIL-10 gene (vIL-10-HSC), ex vivo; vIL-10-HSC were injected (4-6 x 10(6) cells intravenously) into lethally (9.5 Gy) or sublethally (4 Gy) irradiated CBA/J mice. Six weeks after vIL-10-HSC administration, vascular heterotopic heart (C57BL/6) transplantation was performed. Ex vivo, the vIL-10-HSC produced 5.4 +/- 0.5 ng of vIL-10 protein/2 x 10(5) cells per 24 hr. In vivo, serum vIL-10 production was 187 +/- 205 pg/ml during 3-10 weeks after vIL-10-HSC administration. Cardiac allograft survival was prolonged (p < 0.004) in lethally (71 +/- 40 days) and sublethally (114 +/- 15 days) irradiated mice that received vIL-10-HSC compared to controls that received unengineered (UE) HSC or vector DNA-engineered HSC (12-16 days). However, secondary skin graft (C57BL/6) survival was not prolonged in cardiac allograft-tolerant animals. In the vIL-10-HSC-administered group, graft histopathology demonstrated mild arteritis/venulitis (grade 0.7) and rejection (grade 1.0). Intragraft expression of costimulatory molecules (B7.1, B7.2), cytokines (IL-2, IL-4, mIL-10, interferon [IFN]-gamma), and inducible nitric oxide synthase (iNOS) molecules was markedly lower in vIL-10-HSC-treated tolerant grafts that survived more than 100 days compared to vector DNA-HSC- or UE-HSC-treated controls. Furthermore, T lymphocytes derived from vIL-10-HSC-treated tolerant recipients demonstrated hyporeactivity to donor antigens in mixed lymphocyte cultures. Administration of autologous vIL-10-engineered HSC prior to organ transplantation prolonged cardiac allograft survival significantly.

Animals↗
PubMed2006

Tissue engineering cartilage with aged articular chondrocytes in vivo.

BACKGROUND: Tissue engineering has the potential to repair cartilage structures in middle-aged and elderly patients using their own "aged" cartilage tissue as a source of reparative chondrocytes. However, most studies on tissue-engineered cartilage have used chondrocytes from postfetal or very young donors. The authors hypothesized that articular chondrocytes isolated from old animals could produce neocartilage in vivo as well as articular chondrocytes from young donors. METHODS: Articular chondrocytes from 8-year-old sheep (old donors) and 3- to 6-month-old sheep (young donors) were isolated. Cells were mixed in fibrin gel polymer at 40 x 10 cells/ml until polymerization. Cell-polymer constructs were implanted into the subcutaneous tissue of nude mice and harvested at 7 and 12 weeks. RESULTS: Samples and native articular cartilage controls were examined histologically and assessed biochemically for total DNA, glycosaminoglycan, and hydroxyproline content. Histological analysis showed that samples made with chondrocytes from old donors accumulated basophilic extracellular matrix and sulfated glycosaminoglycans around the cells in a manner similar to that seen in samples made with chondrocytes from young donors at 7 and 12 weeks. Biochemical analysis revealed that DNA, glycosaminoglycan, and hydroxyproline content increased in chondrocytes from old donors over time in a pattern similar to that seen with chondrocytes from young donors. CONCLUSIONS: This study demonstrates that chondrocytes from old donors can be rejuvenated and can produce neocartilage just as chondrocytes from young donors do when encapsulated in fibrin gel polymer in vivo. This study suggests that middle-aged and elderly patients could benefit from cartilage tissue-engineering repair using their own "aged" articular cartilage as a source of reparative chondrocytes.

Age Factors↗
PubMed2005

Towards therapeutic applications of engineered zinc finger proteins.

It has long been the goal of molecular biologists to design DNA-binding proteins for the specific control of gene expression. The zinc finger design is ideally suited for such purposes, discriminating between closely related sequences both in vitro and in vivo. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, zinc fingers do not and so can be linked linearly in tandem to recognize DNA sequences of different lengths, with high fidelity. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA. By fusing zinc finger peptides to repression or activation domains, genes can be selectively targeted and switched off and on. Several recent applications of such engineered zinc finger proteins (ZFPs) are described, including the activation of vascular endothelial growth factor (VEGF) in a human cell line and an animal model. Clinical trials have recently begun on using VEGF-activating ZFPs to treat human peripheral arterial disease, by stimulating vascular growth. Also in progress are pre-clinical studies using ZFPs to target the defective genes in two monogenic disorders, SCID and SCA. The aim is to replace them in each case by a correct copy from an extrachromosomal DNA donor by means of homologous recombination. Promising results are reported.

Animals↗
PubMed2006

Molecularly cloned SHIV-1157ipd3N4: a highly replication- competent, mucosally transmissible R5 simian-human immunodeficiency virus encoding HIV clade C Env.

Human immunodeficiency virus type 1 (HIV-1) clade C causes >50% of all HIV infections worldwide, and an estimated 90% of all transmissions occur mucosally with R5 strains. A pathogenic R5 simian-human immunodeficiency virus (SHIV) encoding HIV clade C env is highly desirable to evaluate candidate AIDS vaccines in nonhuman primates. To this end, we generated SHIV-1157i, a molecular clone from a Zambian infant isolate that carries HIV clade C env. SHIV-1157i was adapted by serial passage in five monkeys, three of which developed peripheral CD4(+) T-cell depletion. After the first inoculated monkey developed AIDS at week 137 postinoculation, transfer of its infected blood to a naïve animal induced memory T-cell depletion and thrombocytopenia within 3 months in the recipient. In parallel, genomic DNA from the blood donor was amplified to generate the late proviral clone SHIV-1157ipd3. To increase the replicative capacity of SHIV-1157ipd3, an extra NF-kappaB binding site was engineered into its 3' long terminal repeat, giving rise to SHIV-1157ipd3N4. This virus was exclusively R5 tropic and replicated more potently in rhesus peripheral blood mononuclear cells than SHIV-1157ipd3 in the presence of tumor necrosis factor alpha. Rhesus macaques of Indian and Chinese origin were next inoculated intrarectally with SHIV-1157ipd3N4; this virus replicated vigorously in both sets of monkeys. We conclude that SHIV-1157ipd3N4 is a highly replication-competent, mucosally transmissible R5 SHIV that represents a valuable tool to test candidate AIDS vaccines targeting HIV-1 clade C Env.

Administration, Rectal↗
PubMed1992

Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c.

The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine] (bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) x 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Enumeration of Tn5 mutant bacteria in soil by using a most- probable-number-DNA hybridization procedure and antibiotic resistance.

Investigations were made into the utility of DNA hybridization in conjunction with a microdilution most-probable-number procedure for the enumeration of Rhizobium spp. and Pseudomonas putida in soil. Isolates of Rhizobium spp. and P. putida carrying the transposon Tn5 were added to sterile and nonsterile Burbank sandy loam soil and enumerated over time. Soil populations of rhizobia were enumerated by colony hybridization, most-probable-number-DNA hybridization procedure, plate counts, plant infectivity most probable number, and fluorescent antibody counts. Population values compared well for all methods at 5 and 30 days after the addition of cells, although the fluorescent antibody method tended to overestimate the viable population. In nonsterile soil, most-probable-number-DNA hybridization procedure enumerated as few as 10 P. putida Tn5 cells g of soil-1 and 100 R. leguminosarum bv. phaseoli Tn5 cells g of soil-1 and should have utility for following the fate of genetically engineered microorganisms released to the environment. Among the Kmr isolates containing Tn5, approximately 5% gave a dark, more intense autoradiograph when probed with 32P-labeled pGS9 DNA, which facilitated their detection in soil. Hybridization with a pCU101 probe (pGS9 without Tn5) indicated that donor plasmid sequences were being maintained in the bacterial chromosome. Transposon-associated antibiotic resistance was also utilized as a phenotypic marker. Tn5 vector-integrate mutants were successfully enumerated at low populations (10 to 100 cells g of soil-1) in soil by both phenotypic (Kmr) and genotypic (DNA probe) analysis. However, determination of the stability of Tn5 or Tn5 and vector sequences in the bacteria is necessary.

Bacteriological Techniques↗
PubMed1995

Retrovirus-mediated transduction of an engineered intron-containing purine nucleoside phosphorylase gene.

We constructed and tested several retroviral vectors containing abbreviated purine nucleoside phosphorylase (PNP) genes in the reverse orientation, a strategy compatible with transduction of intron-containing genes. We observed two types of deletions in these vectors after one round of replication: (i) Deletions flanked by direct repeats with one copy of the repeat retained in the provirus, presumably resulting from reverse transcriptase slippage during (-) strand DNA synthesis. (ii) Deletions due to fortuitous splice sites in the PNP complementary strand. Two splice donor sites and three splice acceptor sites were identified in a 3.0-kb PNP minigene. We found that the splice donor sites (but not the splice acceptor sites) could be predicted by sequence analysis of the PNP complementary strand. To increase the frequency of intact PNP gene transduction, we introduced sequence modifications: The putative PNP polyadenylation signal and a truncated 117-bp 3' flank were recovered from a rearranged provirus and inserted in place of a 1.2-kb genomic 3' flank. Sequences associated with deletions were eliminated from the PNP 5' untranslated region, and two fortuitous splice donor signals in the complementary strand were inactivated. A retroviral vector LN-PMG11, containing the engineered 2.9-kb PNP minigene in the reverse orientation, was transduced intact in 23% (5/22) of clones after one round of replication and in 87% (20/23) of clones after a second round of replication from two primary virus producer clones. Directed mutagenesis of sequences preventing intact retroviral transduction thus provided a 2.9-kb PNP gene that was transduced intact and expressed at a high level.

Base Sequence↗
PubMed2006

Relevance and safety of telomerase for human tissue engineering.

Tissue engineering holds the promise of replacing damaged or diseased tissues and organs. The use of autologous donor cells is often not feasible because of the limited replicative lifespan of cells, particularly those derived from elderly patients. Proliferative arrest can be overcome by the ectopic expression of telomerase via human telomerase reverse transcriptase (hTERT) gene transfection. To study the efficacy and safety of this potentially valuable technology, we used differentiated vascular smooth muscle cells (SMC) and vascular tissue engineering as a model system. Although we previously demonstrated that vessels engineered with telomerase-expressing SMC had improved mechanics over those grown with control cells, it is critical to assess the phenotypic impact of telomerase expression in donor cells, because telomerase up-regulation is observed in >95% of human malignancies. To study the impact of telomerase in tissue engineering, expression of hTERT was retrovirally induced in SMC from eight elderly patients and one young donor. In hTERT SMC, significant lifespan extension beyond that of control was achieved without population doubling time acceleration. Karyotype changes were seen in both control and hTERT SMC but were not clonal nor representative of cancerous change. hTERT cells also failed to show evidence of neoplastic transformation in functional assays of tumorigenicity. In addition, the impact of donor age on cellular behavior, particularly the synthetic capability of SMC, was not affected by hTERT expression. Hence, this tissue engineering model system highlights the impact of donor age on cellular synthetic function that appears to be independent of lifespan extension by hTERT.

Blood Vessels↗
PubMed1999

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation.

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.

Acinetobacter↗
PubMed1993

Gene transfer from engineered Lactococcus lactis strains to Enterococcus faecalis in the digestive tract of gnotobiotic mice.

The introduction of genetically modified organisms into food products requires an evaluation of the behaviour and the dissemination of foreign genes of such organisms among the human intestinal microflora. The conjugal transfer, both in vitro and in vivo (in mice digestive tract) of DNA from Lactococcus lactis donor strains to an Enterococcus faecalis strain isolated from human faecal flora was studied. We followed the transfer of (1) the self-transmissible plasmid pIL205; (2) two non-self-transmissible but mobilizable plasmids, pIL252 and pIL253; (3) one plasmid, pMS1.5B, integrated into the chromosome of L. lactis. In vitro, the transfer frequency of pIL205 (expressed as the number of transconjugants per donor cell) was 9.6 x 10(-4); mobilization of one of the non-self-transmissible plasmids, pIL253, was observed (4.9 x 10(-7)). In vivo, only transfer of pIL205 and pIL253 occurred, but the frequency was not determined. The transfer of pMS1.5B was not detected in vitro or in vivo.

Animals↗
PubMed2001

Age dependence of cellular properties of human septal cartilage: implications for tissue engineering.

BACKGROUND: The persistent need for cartilage replacement material in head and neck surgery has led to novel cell culture methods developed to engineer cartilage. Currently, there is no consensus on an optimal source of cells for these endeavors. OBJECTIVES: To evaluate human nasal cartilage as a potential source of chondrocytes and to determine the effect of donor age on cellular and proliferation characteristics. SUBJECTS: Nasal cartilage specimens were obtained after reconstructive surgery from 46 patients ranging in age from 15 to 60 years. METHODS: Specimens were weighed and chondrocytes were isolated by digestion in 0.2% collagenase type II for 16 hours. Cells were maintained in primary cultures until confluency, then seeded onto polylactic acid-polyglycolic acid scaffolds. Seeding efficiency was determined by quantification of DNA content of seeded constructs by means of Hoechst dye 33258. Specimen weights, cell yields, cell content, and doubling time were also measured and correlated to donor age. RESULTS: Mean (+/-SD) cartilage mass obtained (648 +/- 229 mg) is higher than from typical biopsy specimens of auricular cartilage, and the cellular characteristics show a higher proliferation rate than auricular chondrocytes. Cell yield increased with age, while doubling time decreased with age in samples from patients ranging from 15 to 60 years old. CONCLUSIONS: The use of nasal septal cartilage as a source of cells for tissue engineering may be valid over a wide range of patient ages. The large tissue yield and consequent cell yield make this tissue a potential starting source of chondrocytes for large-volume tissue-engineered implants.

Adolescent↗
PubMed1999

Comparative analysis of three genetic modifications designed to inhibit human serum-mediated cytolysis.

Hyperacute rejection (HAR) remains a critical immunologic hurdle in the development of xenogeneic organs for human transplantation. Strategies that simultaneously eliminate both natural antibody reactivity and complement activation on the xenogeneic cell surface may be the best approach to achieve clinical application of xenogeneic vascularized organ transplantation. We have developed multiple lines of genetically manipulated mice to evaluate the combination of different genetic approaches aimed at inhibiting antibody and complement-mediated cell lysis. We utilized transgenic mice expressing the human complement inhibitor, CD59, the human 1,2-fucosyltransferase (H-transferase, HT) and the alpha1,3-galactosyltransferase (alpha1,3-GT) knock-out mouse line (Gal KO). Our data show that expression of hCD59 in combination with HT expression or the null phenotype of alpha1,3-GT are equally effective at preventing human serum-mediated cytolysis. Interestingly, the triple combination affords no additional protective effect. Therefore, coexpression of HT and a complement inhibitor is the most immediate strategy to genetically engineer transgenic pigs to be used as xenogeneic donors.

Animals↗
PubMed2001

Human dendritic cells genetically engineered to express a melanoma polyepitope DNA vaccine induce multiple cytotoxic T-cell responses.

PURPOSE: To assess the therapeutic potential of a melanoma polyepitope vaccine in human cells. Polyepitope DNA vaccines encoding T-cell epitopes have been demonstrated in murine systems to generate multiple cytotoxic T-cell responses to different antigens. Here, for the first time we demonstrate the ability of a melanoma polyepitope to stimulate lymphocytes from normal human donors to simultaneously generate multiple antigen-specific responses. EXPERIMENTAL DESIGN: Human dendritic cells (DC), transduced with a melanoma-polyepitope cDNA, were used to activate autologous lymphocytes from naïve donors as an in vitro model of DNA vaccination. Lymphocytes were primed with polyepitope or mock-transduced DC, boosted with peptide, then measured for antigen-specific cytotoxicity. RESULTS: Lymphocytes primed with polyepitope-transduced DC and boosted with peptide generated multiple cytotoxic responses. By contrast lymphocytes primed with mock-transfected DCs and boosted with peptide gave no specific cytotoxicity. However, when lymphocytes were repeatedly stimulated with polyepitope-transduced DCs immunodominance was seen with CTLs being generated to only one epitope, MART(27-35). CONCLUSIONS: We show in a human system that a melanoma polyepitope primes CTL to multiple epitopes. However, repeated stimulation by the polyepitope restricts the response to only the MART1 epitope. Thus, although polyepitope vaccines are an effective way of priming multiple naïve T-cell responses, continual boosting with polyepitope vaccines may, as a result of immunodominance, restrict the CTL. These findings have important implications for the use of DNA polyepitope vaccines in cancer immunotherapy.

Animals↗
PubMed2005

High-frequency homologous recombination in plants mediated by zinc-finger nucleases.

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.

Base Sequence↗
PubMed2005

Highly efficient endogenous human gene correction using designed zinc-finger nucleases.

Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rgamma gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.

Alleles↗
PubMed2005

MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules.

We describe a highly engineered in vivo cloning method, mating-assisted genetically integrated cloning (MAGIC), that facilitates the rapid construction of recombinant DNA molecules. MAGIC uses bacterial mating, in vivo site-specific endonuclease cleavage and homologous recombination to catalyze the transfer of a DNA fragment between a donor vector in one bacterial strain and a recipient plasmid in a separate bacterial strain. Recombination events are genetically selected and result in placement of the gene of interest under the control of new regulatory elements with high efficiency. MAGIC eliminates the need for restriction enzymes, DNA ligases, preparation of DNA and all in vitro manipulations required for subcloning and allows the rapid construction of multiple constructs with minimal effort. We show that MAGIC can generate constructs for expression in multiple organisms. As this new method requires only the simple mixing of bacterial strains, it represents a substantial advance in high-throughput recombinant DNA production that will save time, effort and expense in functional genomics studies.

Amino Acid Sequence↗