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PubMed1995

Expression of biologically active basic fibroblast growth factor by genetically modified rat primary skin fibroblasts.

Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control of FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.

Animals↗
PubMed2004

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures.

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 beta-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T(3)) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H(11) (184 mg/liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

Alkaloids↗
PubMed2000

Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors.

Genetically engineered, neuroattenuated herpes simplex viruses (HSVs) expressing various cytokines can improve survival when used in the treatment of experimental brain tumors. These attenuated viruses have both copies of gamma(1)34.5 deleted. Recently, we demonstrated increased survival of C57BL/6 mice bearing syngeneic GL-261 gliomas when treated with an engineered HSV expressing IL-4, as compared with treatment with the parent construct (gamma(1)34. 5(-)) alone or with a virus expressing IL-10. Herein, we report construction of a conditionally replication-competent mutant expressing both subunits of mIL-12 (M002) and its evaluation in a syngeneic neuroblastoma murine model. IL-12 induces a helper T cell subset type 1 response, which may induce more durable antitumor effects. In vitro studies showed that, when infected with M002, both Vero cells and murine Neuro-2a neuroblastoma cells produced physiologically relevant levels of IL-12 heterodimers, as determined by ELISA. M002 was cytotoxic for Neuro-2a cells and human glioma cell lines U251MG and D54MG. Neurotoxicity studies, as defined by plaque-forming units/LD(50), performed in HSV-1-sensitive A/J strain mice found that M002 was not toxic even at high doses. When evaluated in an intracranial syngeneic neuroblastoma murine model, median survival of M002-treated animals was significantly longer than the median survival of animals treated with R3659, the parent gamma(1)34.5(-) mutant lacking any cytokine gene insert. Immunohistochemical analysis of M002-treated tumors identified a pronounced influx of CD4(+) T cells and macrophages as well as CD8(+) cells when compared with an analysis of R3659-treated tumors. We conclude that M002 produced a survival benefit via oncolytic effects combined with immunologic effects meditated by helper T cells of subset type 1.

Animals↗
PubMed2005

Transgenic engineering of male-specific muscular hypertrophy.

Using a two-step procedure involving insertional gene targeting and recombinase-mediated cassette exchange in ES cells, we have produced two lines of transgenic mice expressing a dominant-negative latency-associated myostatin propeptide under control of the myosin light chain 1F promoter and 1/3 enhancer from the TSPY locus on the Y chromosome. Males of the corresponding lines are characterized by a 5-20% increase in skeletal muscle mass. This experiment demonstrates the feasibility of a more efficient cattle production system combining superior beef production abilities for bulls and dairy abilities for cows.

Animals↗
PubMed2005

An alternative strategy for sustainable pest resistance in genetically enhanced crops.

Bacillus thuringiensis (Bt) crystal protein genes encode insecticidal delta-endotoxins that are widely used for the development of insect-resistant crops. In this article, we describe an alternative transgenic strategy that has the potential to generate broader and more sustainable levels of resistance against insect pests. Our strategy involves engineering plants with a fusion protein combining the delta-endotoxin Cry1Ac with the galactose-binding domain of the nontoxic ricin B-chain (RB). This fusion, designated BtRB, provides the toxin with additional, binding domains, thus increasing the potential number of interactions at the molecular level in target insects. Transgenic rice and maize plants engineered to express the fusion protein were significantly more toxic in insect bioassays than those containing the Bt gene alone. They were also resistant to a wider range of insects, including important pests that are not normally susceptible to Bt toxins. The potential impact of fusion genes such as BtRB in terms of crop improvement, resistance sustainability, and biosafety is discussed.

Analysis of Variance↗
PubMed2006

Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system.

Plague is still an endemic disease in different regions of the world. Increasing reports of incidence, the discovery of antibiotic resistance strains, and concern about a potential use of the causative bacteria Yersinia pestis as an agent of biological warfare have highlighted the need for a safe, efficacious, and rapidly producible vaccine. The use of F1 and V antigens and the derived protein fusion F1-V has shown great potential as a protective vaccine in animal studies. Plants have been extensively studied for the production of pharmaceutical proteins as an inexpensive and scalable alternative to common expression systems. In the current study the recombinant plague antigens F1, V, and fusion protein F1-V were produced by transient expression in Nicotiana benthamiana by using a deconstructed tobacco mosaic virus-based system that allowed very rapid and extremely high levels of expression. All of the plant-derived purified antigens, administered s.c. to guinea pigs, generated systemic immune responses and provided protection against an aerosol challenge of virulent Y. pestis.

Aerosols↗
PubMed2006

Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol.

Global cottonseed production can potentially provide the protein requirements for half a billion people per year; however, it is woefully underutilized because of the presence of toxic gossypol within seed glands. Therefore, elimination of gossypol from cottonseed has been a long-standing goal of geneticists. Attempts were made to meet this objective by developing so-called "glandless cotton" in the 1950s by conventional breeding techniques; however, the glandless varieties were commercially unviable because of the increased susceptibility of the plant to insect pests due to the systemic absence of glands that contain gossypol and other protective terpenoids. Thus, the promise of cottonseed in contributing to the food requirements of the burgeoning world population remained unfulfilled. We have successfully used RNAi to disrupt gossypol biosynthesis in cottonseed tissue by interfering with the expression of the delta-cadinene synthase gene during seed development. We demonstrate that it is possible to significantly reduce cottonseed-gossypol levels in a stable and heritable manner. Results from enzyme activity and molecular analyses on developing transgenic embryos were consistent with the observed phenotype in the mature seeds. Most relevant, the levels of gossypol and related terpenoids in the foliage and floral parts were not diminished, and thus their potential function in plant defense against insects and diseases remained untouched. These results illustrate that a targeted genetic modification, applied to an underutilized agricultural byproduct, provides a mechanism to open up a new source of nutrition for hundreds of millions of people.

Diet↗
PubMed2000

A phage integrase directs efficient site-specific integration in human cells.

The integrase from the Streptomyces phage phiC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the phiC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

Bacteriophages↗
PubMed1982

Expression of the structural proteins of Semliki Forest virus from cloned cDNA microinjected into the nucleus of baby hamster kidney cells.

The three structural proteins of Semliki Forest virus--i.e., the capsid, p62, and E1 proteins--were expressed in baby hamster kidney cells from cloned DNA transcribed from the virus-specific 4.1-kilobase mRNA. The cDNA was engineered into an expression vector developed by others [Mulligan, R. C. & Berg, P. (1980) Science 209, 1422--1427] downstream from the simian virus 40 early promoter and was introduced into cell nuclei by microneedle injection. Immunofluorescence analysis of injected cells showed that the capsid protein was located in the cell cytoplasm, whereas the membrane proteins were associated with cellular membranes. The p62 protein was shown to be transported from the rough endoplasmic reticulum to the plasma membrane, whereas the E1 protein remained in the rough endoplasmic reticulum.

Animals↗
PubMed1984

Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis.

We have developed a rapid and general technique for purification of a protein encoded by a cistron contained in a recombinant DNA clone. The technique consists of fusing the target cistron DNA in the correct reading frame to a marker cistron via a piece of DNA that codes for a linker peptide. The target cistron in the example presented here is the replication initiator cistron of the plasmid R6K. The linker is a DNA fragment encoding 60 amino acids from the triple helical region of chicken pro alpha-2 collagen, and the marker cistron encodes the beta-galactosidase protein of Escherichia coli. The tripartite hybrid protein was rapidly purified by selective binding to and elution from a beta-galactosidase specific-affinity column. The hybrid protein was then digested with a purified microbial collagenase to cleave the linker, and high-pressure liquid chromatography allowed the rapid isolation of the target protein from the marker protein. Using this technique, we have purified the highly labile R6K replication initiator to homogeneity, and we have resolved the protein into NH2-terminal and COOH-terminal segments. We have further shown, by in vitro binding, that the COOH-terminal segment has at least one DNA-binding domain. The domain binds to the same restriction fragments of the R6K chromosome as the intact or beta-galactosidase-tagged initiator protein.

Bacteriophage lambda↗
PubMed1985

Transfer of genes into hematopoietic cells using recombinant DNA viruses.

The ability of recombinant DNA viruses to transfer genes into hematopoietic cells has been explored. A recombinant simian virus 40 (SV40) in which the early region had been replaced with the chloramphenicol acetyltransferase (CAT) gene driven by the promoter from Rous sarcoma virus (RSV), was constructed. This virus transferred the CAT gene more efficiently into mouse and human bone marrow cells and into the K562, MEL, and WEHI hematopoietic tissue culture cell lines, than the classical calcium phosphate DNA transfer procedure, as shown by assay for CAT activity 48 hr after infection. Recombinant SV40 virions were also shown to be capable of stably transforming Chinese hamster ovary cells by use of an early region recombinant containing the methotrexate-resistant dihydrofolate reductase (DHFR) gene driven by the RSV promoter. The entire DHFR transcriptional unit could be detected in the genome of transformed cells that were also shown to be resistant to methotrexate. A recombinant adenovirus stock containing the neomycin-resistance gene driven by the SV40 early promoter was used to infect the K562 and MEL hematopoietic cell lines to resistance to the antibiotic G418. Transformation frequency was 10- to 100-fold higher than that obtained with calcium phosphate-precipitated DNA. Most or all of the recombinant adenovirus genome was integrated as 1-3 copies in the transformed cells. These studies show the feasibility of using DNA viruses for introduction of new genetic material into hematopoietic cells.

Acetyltransferases↗
PubMed1985

Expression of phaseolin cDNA genes in yeast under control of natural plant DNA sequences.

We have constructed a strain of Saccharomyces cerevisiae that expresses two different members of the multigene family encoding phaseolin, the major seed storage glycoprotein from the French bean, Phaseolus vulgaris. Yeast vector plasmids have been engineered to include a Phaseolus DNA segment that contains the natural 5' and 3' plant genomic regulatory sequences flanking a cDNA copy of the protein-encoding region. Characterization of phaseolin transcripts isolated from transformed yeast cells revealed the presence of two classes of polyadenylylated RNA, approximately 1400 and 1800 bases, which initiate and terminate in plant DNA sequences. Protein extracts from transformants contain phaseolin-immunoreactive proteins similar in size to those isolated from plant tissue. These polypeptides are glycosylated in yeast and their molecular weights are consistent with the possibility that the phaseolin signal peptide has been cleaved to form the mature protein.

Journal Article↗
PubMed1986

Introduction of genes into preimplantation mouse embryos by use of a defective recombinant retrovirus.

Two-cell and four-cell preimplantation mouse embryos were cocultured in vitro with fibroblasts producing the recombinant retrovirus M-MuLV-neo. No wildtype helper virus was detected in these cultures. Of the embryos that survived the in vitro cultivation and the reimplantation into foster mothers, 2 of 15 that were tested contained the proviral genome. The provirus integrated as a single copy at a unique site. We estimate that approximately equal to 20% of the cells in each of the two transgenic fetuses contained the provirus.

Animals↗
PubMed1986

Rapid mapping of antigenic coding regions and constructing insertion mutations in yeast genes by mini-Tn10 "transplason" mutagenesis.

A "transplason" mutagenesis procedure was developed for the dual purposes of low resolution mapping of antigenic coding regions (using transposons) and constructing insertion mutations in yeast genes (by transplacement). Mini-Tn10 transposon derivatives containing both Escherichia coli and yeast selectable markers have been constructed. These elements are used to mutagenize lambda gt11 clones that express foreign antigens in E. coli. The transposition events are first selected in E. coli, and the effect of these insertions on antigen expression is used to locate the antigenic coding regions on the cloned DNA. Insertion mutations located within a desired yeast sequence are then substituted for the genomic copies by one-step gene transplacement. This provides a powerful method for rapidly mapping antigenic coding sequences of cloned genes and inactivating these genes in yeast to help determine their function. Several examples using this technique are presented.

Antibodies↗
PubMed1986

A genomic clone encoding the alpha chain of the OKM1, LFA-1, and platelet glycoprotein IIb-IIIa molecules.

LFA-1, an antigen involved in cytolytic T lymphocyte-mediated killing, and Mac-1, the receptor for complement component C3bi, constitute a family of structurally and functionally related cell surface glycoproteins involved in cellular interactions. In both mouse and man, Mac-1 (OKM1) and LFA-1 share a common 95-kDa beta subunit but are distinguished by their alpha chains, which have different cellular distributions, apparent molecular masses (165 and 177 kDa, respectively), and peptide maps. We report the isolation of a genomic clone from a human genomic library that on transfection into mouse fibroblasts produced a molecule(s) reactive with monoclonal antibodies to OKM1, to LFA-1, and to platelet glycoprotein IIb-IIIa. This gene was cloned by several cycles of transfection of L cells with a human genomic library cloned in lambda phage Charon 4A and subsequent "rescue" of the lambda phage. Transfection with the purified recombinant lambda DNA yielded a transfectant that expressed the three human alpha chains of OKM1, LFA-1, and glycoprotein IIb-IIIa, presumably in association with the murine beta chain.

Antibodies, Monoclonal↗
PubMed1986

Retrovirus-mediated transfer of human adenosine deaminase gene sequences into cells in culture and into murine hematopoietic cells in vivo.

Deficiency of the enzyme adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4; ADA) leads to severe combined immunodeficiency, a disorder that potentially could be corrected by gene transfer into hematopoietic cells. We have constructed retroviruses containing human ADA cDNA and a dominant selectable marker, a mutated dihydrofolate reductase gene (DHFR*) encoding methotrexate resistance. Human ADA cDNA was inserted alone (DHFR*-ADA) or with a simian virus 40 (SV40) promoter (DHFR*-SVADA). Although NIH 3T3 cells infected with either construct produced human ADA activity, substantially greater levels were attained with DHFR*-SVADA. Infection of murine lymphoid cells in culture with DHFR*-SVADA led to expression of human enzyme at a level well above the mouse endogenous level. ADA activity was also increased after infection of a human ADA-deficient B-cell line. Lethally irradiated mice that were reconstituted with syngeneic marrow infected with the DHFR*-SVADA virus contained unrearranged, integrated proviral DNA in total spleen DNA or in spleen hematopoietic stem cell (CFU-S)-derived colonies. Nevertheless, no human ADA was detectable. RNA analysis showed relatively low and variable expression from the retroviral long terminal repeat, and no detectable expression from the internal SV40 promoter. These data suggest that intrinsic biologic differences exist between cultured cells and CFU-S in vivo.

Adenosine Deaminase↗
PubMed1987

pH-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse.

A plasmid containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of a mammalian cAMP-regulated promoter was entrapped in H-2Kk antibody-coated liposomes composed of dioleoyl phosphatidylethanolamine, cholesterol, and oleic acid (pH-sensitive immunoliposomes). The entrapped or free DNA was injected intraperitoneally into immunodeficient (nude) BALB/c mice bearing ascites tumor generated by H-2Kk-positive RDM-4 lymphoma cells. About 20% of the injected immunoliposomes were taken up by the target RDM-4 cells. Uptake was much less when liposomes without antibody were used. The presence of the targeting antibody on liposomes also significantly decreased the nonspecific uptake of liposomes by the spleen. Significant CAT enzyme activity was detected in RDM-4 cells from mice treated with DNA entrapped in the pH-sensitive immunoliposomes. Furthermore, CAT expression in RDM-4 cells was under the control of cAMP, as only the cells from mice injected with 8-bromo-cAMP and 3-isobutyl-1-methylxanthine showed CAT activity. CAT activity in liver and spleen was much lower (by factors of 12 and 5, respectively) than in the RDM-4 cells, and the activities in these reticuloendothelial organs were not regulated by cAMP. CAT activity in RDM-4 cells from mice injected with DNA entrapped in pH-insensitive immunoliposomes (containing phosphatidylcholine in place of phosphatidylethanolamine) was approximately one-fourth that in RDM-4 cells from mice injected with pH-sensitive immunoliposomes, indicating the superior delivery efficiency of the pH-sensitive liposomes. These results are discussed in terms of the DNA-carrier potential of immunoliposomes in therapy of cancer and genetic diseases.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.

Adenosine Deaminase↗