Expression Of Foreign Gene Products Research Articles

Baculovirus expression systems for production of recombinant proteins in insect and mammalian cells.

Baculovirus vector systems are extensively used for the expression of foreign gene products in insect and mammalian cells. New advances increase the possibilities and applications of the baculovirus expression system, which makes it possible to express multiple genes simultaneously within a single infected insect cell and to obtain multimeric proteins functionally similar to their natural analogs. Recombinant viruses with expression cassettes active in mammalian cells are used to deliver and express genes in mammalian cells in vitro and in vivo. Further improvement of the baculovirus expression system and its adaptation to specific target cells can open up a wide variety of applications. The review considers recent achievements in the use of modified baculoviruses to express recombinant proteins in eukaryotic cells, advantages and drawbacks of the baculovirus expression system, and ways to optimize the expression of recombinant proteins in both insect and mammalian cell lines.

Comparative Studies of Lepidopteran Baculovirus-Specific Protein FP25K: Development of a Novel Bombyx mori Nucleopolyhedrovirus-Based Vector with a Modified fp25K Gene

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.

Induction of T cell tolerance to a protein expressed in the cytoplasm through retroviral-mediated gene transfer.

Host immune responses to foreign gene products have been shown to lead to the elimination of genetically modified cells, and are a major barrier to successful therapeutic gene therapy. We have shown that immunological tolerance to retrovirally transduced cell surface proteins can be induced by expressing the gene encoding these products in bone marrow derived cells. Here, we investigate if expression of foreign gene products in bone marrow derived cells can be used to induce tolerance to cytoplasmic proteins. Balb/c mice were reconstituted with syngeneic bone marrow cells transduced with retrovirus carrying the gene encoding enhanced green fluorescent protein (eGFP), or mock-transduced bone marrow cells. After reconstitution, mice were immunized with cells expressing eGFP, and T cells were tested for the ability to kill eGFP-expressing targets in in vitro cytotoxic T lymphocyte (CTL) assays. T cells from Balb/c mice reconstituted with mock-transduced bone marrow were able to kill target cells expressing eGFP. In contrast, T cells from mice reconstituted with eGFP-transduced bone marrow were unable to kill targets expressing eGFP. In addition, we observed that T cell responses to eGFP in C57BL/6 mice were minimal even under highly immunogenic conditions. These data suggest that expression of foreign gene products in bone marrow derived cells is capable of inducing T cell tolerance to proteins expressed exclusively in the cytoplasm.

Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor.

Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells. We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 microgram/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes: (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency. Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of foreign genes and inhibition of specific nucleases may facilitate transfection.

A peroxidase gene promoter induced by phytopathogens and methyl jasmonate in transgenic plants.

The expression of two closely related peroxidase isogenes, Shpx6a and Shpx6b, of the legume Stylosanthes humilis was studied using isogene-specific reverse transcriptase PCR techniques. Results indicated that transcripts of both genes were rapidly induced following inoculation with the fungal pathogen Colletotrichum gloeosporioides, wounding and treatment with the defense regulator methyl jasmonate (MeJA). In contrast treatment of leaves of S. humilis with abscisic acid (ABA) and salicylic acid (SA) did not induce transcripts of either isogene. A genomic clone containing the Shpx6b gene was isolated and 594 bp of 5' sequence upstream of the translation start was fused in frame to the coding region of the uidA reporter gene and introduced into tobacco. Expression from the Shpx6b promoter in transgenic plants was determined by histochemical staining and quantitative assays of beta-glucuronidase (GUS). In transgenic tobacco, GUS expression was detected in cotyledons, vascular cells of young leaves, anthers, pollen, and the stigma and style. Wounding of the tobacco plants produced very localized GUS staining. Much more extensive staining for GUS was observed following inoculation of tobacco leaves with conidia of the fungal pathogen Cercospora nicotianae and the inoculation of wound sites with mycelium of the Oomycete pathogen Phytophthora parasitica var. nicotianae. Treatment of mature leaves with methyl jasmonate induced GUS activity while treatment with ABA, SA, and H2O2 had no effect. A similar strong induction of GUS activity was measured in young transgenic seedlings germinated on MeJA while some, but much weaker, induction of GUS activity was observed in seedlings treated with SA. The sequence of the promoter contained motifs homologous to putative cis elements in other plant genes responsive to MeJA. The Shpx6b gene is the first plant peroxidase gene shown to be induced by both microbial pathogens and MeJA and its promoter will be useful for investigations of signaling processes during fungal infection and for the expression of foreign gene products at infection sites.

Effect of canarypox virus (ALVAC)-mediated cytokine expression on murine prostate tumor growth.

Canarypox virus, ALVAC, does not replicate in infected mammalian cells and has potential as a vector for gene therapy in the treatment of cancer. Recombinant viruses carrying DNA sequences encoding interleukin 2 (ALVAC-IL-2), interferon gamma (ALVAC-IFN gamma), tumor necrosis factor-alpha (ALVAC-TNF-alpha), or the co-stimulatory molecule B7-1 (ALVAC-B7-1) were investigated as agents for the treatment of a newly defined mouse prostate tumor model. RM-1 mouse prostate cancer cells, which are syngeneic (i.e., same genetic background) to C57BL/6 mice, were used. The expression of foreign gene products in vitro in infected RM-1 cells was measured by immunoprecipitation, bioassay, or flow cytometry. The effects of foreign gene product expression on RM-1 tumor cell growth in C57BL/6 mice were measured after subcutaneous injection (in the back) of 5 x 10(5) uninfected or infected cells; measurements included determinations of time to a measurable tumor size, tumor size as a function of time, and survival. The induction of protective immunity by uninfected and infected RM-1 cells was tested by injection of lethally irradiated (70 Gy) cells and subsequent challenge with uninfected cells. The generation of cytotoxic T cells was monitored by use of a 51Cr release assay. Severe combined immunodeficient (SCID) mice were used to determine whether T or B lymphocytes were involved in ALVAC vector-mediated antitumor responses. Data were analyzed by use of Pearson's modification of the chi-squared test and Kaplan-Meier survival methods. Reported P values are two-sided. The level of foreign gene product expression in ALVAC-infected RM-1 cells was dependent on the multiplicity of virus infection used; a multiplicity of five viruses per infected cell was chosen for subsequent experiments. RM-1 tumor growth in C57BL/6 mice was not affected by tumor cell expression of IL-2 alone, IFN gamma alone, or B7-1 alone; however, expression of TNF-alpha alone significantly delayed tumor growth at early time points (compared with parental ALVAC-infected tumors, P = .0001 at day 21 and P = .037 at day 28). Tumor cell expression of both TNF-alpha and IL-2 completely inhibited tumor growth in 60%-100% of treated mice. No protection against subsequent tumor challenge was detected in mice previously exposed to RM-1 cells expressing both TNF-alpha and IL-2. Cytotoxic T-lymphocyte activity toward RM-1 cells was not observed in C57BL/6 mice that rejected tumors. Tumor cell expression of TNF-alpha and IL-2 also resulted in tumor growth inhibition in SCID mice. RM-1 mouse prostate cancer cells are readily infected by ALVAC vectors, and foreign gene products are efficiently expressed. Inhibition of RM-1 tumor growth by tumor cell expression of TNF-alpha and IL-2 appears to involve nonspecific antitumor activity.

Activities of CaMV 35S andnospromoters in pollen: implications for field release of transgenic plants

The expression of foreign genes in pollen may pose potential problems in the field release of transgenic plants, since pollen represents a route whereby foreign genes and their products may escape into the wider environment. The possible risks posed by cross-hybridization with wild relatives have been extensively explored, but problems that may arise due to the expression of foreign gene products in pollen have not been so widely studied. The activities of the CaMV 35S and nos promoters in pollen in populations of stably transformed plants and in transient expression analysis are described. These promoters are commonly used in all areas of plant molecular biology research and their expression patterns will be of interest to those involved in field release studies. The results show that both promoters had no detectable pollen activity in Arabidopsis, but both showed activity in tobacco pollen. The CaMV 35S-gus gene fusion showed heritable expression levels in tobacco pollen of up to a maximum of 64.6 pmol 4-MU min−1 mg −1 total protein. nos promoter activity in transgenic tobacco pollen was highly variable, with GUS activities ranging from undetectable levels up to 2561 pmol 4-MU min−1 mg−1 total protein within the transgenic population. Histochemical staining of anther sections from 10–12 mm buds revealed that the CaMV 35S promoter had some activity in the vascular bundle, stomium and tapetum, while GUS expression from the nos promoter in sporophytic tissues was confined entirely to the stomium.

Construction of recombinant human type 5 adenoviruses expressing rodent IL-6 genes. An approach to investigate in vivo cytokine function.

The majority of biologic functions assigned to cytokines have been characterized by in vitro assay systems which may not necessarily reflect cytokine roles in vivo. Recently, recombinant virus approaches have allowed tissue-specific expression of foreign gene products in experimental animal models. We have constructed recombinant human type 5 adenoviruses, deficient in the E3 region of the genome, with incorporated rodent IL-6 cDNA that express significant levels of biologically active IL-6 on infection both in vitro and in vivo. After i.p. injection, the liver, spleen, and peritoneum appear to be primary sites of expression, whereas the lung and bronchus are the main sites of expression after intratracheal instillation. Injection i.p. of BALB/c mice with the murine rIL-6 virus causes an increase in serum levels of bioactive IL-6 for up to 6 days post-infection, whereas similar changes are not seen in animals infected with control viruses. Coincident with enhanced plasma levels of IL-6, we detect raised serum levels of hepatic-derived acute phase proteins. Associated with the expression of IL-6 in the liver and spleen, at 7 days we note a fourfold splenomegaly with expansion of B and T cell compartments, as well as the presence of lymphoid aggregates in the liver. These morphologic changes had resolved by 16 days. Our findings demonstrate that recombinant human type 5 adenoviruses expressing cDNA for various cytokines could be used as a transient pseudo-transgenic animal model to investigate the biologic function of cytokines in vivo.

Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells.

Baculoviruses are currently used as vectors for the transient high-level expression of foreign gene products in insect cells. In this study, we demonstrate that baculoviruses can also be made to continuously express a foreign gene product by using the promoter from IE1, an immediate early viral gene, to produce stably-transformed insect cells. This approach gave levels of foreign gene expression lower than those usually obtained with the lytic baculovirus expression vector system. Expression, however, was continuous and stable, and a complex human glycoprotein (tissue plasminogen activator) was processed more efficiently. We conclude that stable transformation is a feasible approach for baculovirus-mediated foreign gene expression in lepidopteran cells, particularly for products that are relatively poorly-expressed and/or processed in lytically infected cells.

Isolation, sequence, and expression of a human keratin K5 gene: transcriptional regulation of keratins and insights into pairwise control.

The mitotically active basal layers of most stratified squamous epithelia express 10 to 30% of their total protein as keratin. The two keratins specifically expressed in these cells are the type II keratin K5 (58 kilodaltons) and its corresponding partner, type I keratin K14 (50 kilodaltons), both of which are essential for the formation of 8-nm filaments. Dissecting the molecular mechanisms underlying the coordinate regulation of the two keratins is an important first step in understanding epidermal differentiation and in designing promoters that will enable delivery and expression of foreign gene products in stratified squamous epithelia, e.g., skin. Previously, we reported the sequence of the gene encoding human K14 (D. Marchuk, S. McCrohon, and E. Fuchs, Cell 39:491-498, 1984; Marchuk et al., Proc. Natl. Acad. Sci. USA 82:1609-1613, 1985). We have now isolated and characterized the gene encoding human K5. The sequence of the coding portion of this gene matched perfectly with that of a partial K5 cDNA sequence obtained from a cultured human epidermal library (R. Lersch and E. Fuchs, Mol. Cell. Biol. 8:486-493, 1988), and gene transfection studies indicated that the gene is functional. Nuclear runoff experiments demonstrated that the K5 and K14 genes were both transcribed at dramatically higher levels in cultured human epidermal cells than in fibroblasts, indicating that at least part of the regulation of the expression of this keratin pair is at the transcriptional level. When the K5 gene was transfected transiently into NIH 3T3 fibroblasts, foreign expression of the gene caused the appearance of endogenous mouse K14 and the subsequent formation of a keratin filament array in the cells. In this case, transcriptional changes did not appear to be involved in the regulation, suggesting that there may be multiple control mechanisms underlying the pairwise expression of keratins.

Isolation, Sequence, and Expression of a Human Keratin K5 Gene: Transcriptional Regulation of Keratins and Insights into Pairwise Control

The mitotically active basal layers of most stratified squamous epithelia express 10 to 30% of their total protein as keratin. The two keratins specifically expressed in these cells are the type II keratin K5 (58 kilodaltons) and its corresponding partner, type I keratin K14 (50 kilodaltons), both of which are essential for the formation of 8-nm filaments. Dissecting the molecular mechanisms underlying the coordinate regulation of the two keratins is an important first step in understanding epidermal differentiation and in designing promoters that will enable delivery and expression of foreign gene products in stratified squamous epithelia, e.g., skin. Previously, we reported the sequence of the gene encoding human K14 (D. Marchuk, S. McCrohon, and E. Fuchs, Cell 39:491-498, 1984; Marchuk et al., Proc. Natl. Acad. Sci. USA 82:1609-1613, 1985). We have now isolated and characterized the gene encoding human K5. The sequence of the coding portion of this gene matched perfectly with that of a partial K5 cDNA sequence obtained from a cultured human epidermal library (R. Lersch and E. Fuchs, Mol. Cell. Biol. 8:486-493, 1988), and gene transfection studies indicated that the gene is functional. Nuclear runoff experiments demonstrated that the K5 and K14 genes were both transcribed at dramatically higher levels in cultured human epidermal cells than in fibroblasts, indicating that at least part of the regulation of the expression of this keratin pair is at the transcriptional level. When the K5 gene was transfected transiently into NIH 3T3 fibroblasts, foreign expression of the gene caused the appearance of endogenous mouse K14 and the subsequent formation of a keratin filament array in the cells. In this case, transcriptional changes did not appear to be involved in the regulation, suggesting that there may be multiple control mechanisms underlying the pairwise expression of keratins.

Regulated expression at high copy number allows production of a growth-inhibitory oncogene product in Drosophila Schneider cells.

The Drosophila metallothionein promoter (Mtn) was used to obtain efficient, regulated expression of foreign gene products inserted in high copy numbers into Drosophila melanogaster Schneider 2 cells. An expression unit comprised of a reporter gene [Escherichia coli galactokinase (galK)] fused to the Mtn promoter was stably introduced into Schneider 2 cells in up to several hundred copies per cell in a single transfection-selection event. This system contrasts dramatically with other eukaryotic systems that permit only a few copies of a gene to be stably inserted in a single transfection-selection event. The transfected Drosophila S2 cell lines expressed high levels of both galK mRNA and protein in response to metal induction. Most important, and in contrast to mammalian cells, expression remained fully regulated even at high copy number, with low basal expression maintained in the absence of inducer. This regulated system was used to obtain efficient expression in Drosophila cells of an otherwise lethal or growth-inhibitory gene product, the human H-ras oncogene. The ability to obtain regulated high-level expression of potentially lethal foreign proteins is unique to the Drosophila cell system.

Insertion and deletion mutagenesis of the human cytomegalovirus genome.

Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. We have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major beta gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this beta gene within the L-component repeats of CMV DNA. We observed high-level expression of beta-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside, we generated random endpoint deletion mutants. Analysis of these mutants revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, we have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.