Eukaryotic and prokaryotic signal peptides direct secretion of a bacterial endoglucanase by mammalian cells

It is well established that hydrophobic signal sequences direct proteins into or across the endoplasmic reticulum membrane in eukaryotes and cell membranes in prokaryotes. Although it is recognized that eukaryote proteins are efficiently secreted by bacterial systems, the export of bacterial proteins by eukaryotes has received little attention. To investigate membrane translocation of bacterial proteins by mammalian cells, the secretion of a bacterial endoglucanase (endoglucanase E) from stably transfected Chinese hamster ovary cells has been examined. We report that a functional endoglucanase is secreted when fused to prokaryote or eukaryote signal peptides. Furthermore, the endoglucanase was post-translationally modified before secretion. Data presented in this paper suggest that secretion of bacterial proteins by eukaryote cells may be a general phenomenon and infer that there are no specific requirements with respect to the origin of the signal sequences.     

Expression of a bacterial repair gene in mammalian cells

The coding sequence of the uvrA gene from Escherichia coli has been fused to the early promoter, enhancer and origin of replication of the simian virus SV40, and was supplemented with splicing and polyadenylation sites arising from the same virus. Introduction of this hybrid gene into simian cos-1 cells results in the synthesis of a full length UvrA protein (114 kD) which has retained its ability to bind to single-stranded DNA.     

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.     

Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.     

Optimization of genetic constructs for high-level expression of the darbepoetin gene in mammalian cells

Genetic constructs were designed in order to optimize darbepoetin production in CHO cells. They are characterized by a higher level of structural optimization of the darbepoetin gene, a higher gene dose, and the selection of promoter elements that have never been used before for this purpose. A transient transfection of CHO cells by the obtained constructs was performed. It was shown that each of the variable factors in the constructs influenced darbepoetin gene expression. A construct containing a doubled dose of the darbepoetin synthetic gene with optimized codon composition under the control of the CMV-EF1α chimerical promoter was proved to be the most efficient.     

Magnetic resonance-based visualization of gene expression in mammalian cells using a bacterial polyphosphate kinase reporter gene

Gene expression reporter systems, in which a promoter of interest is cloned upstream of a readily assayed reporter gene, have been developed and used extensively to study gene expression in prokaryotes and eukaryotes. Unfortunately, most of these systems cannot be used to assay gene expression in nonsuperficial tissues in living organisms. This study examines a novel reporter gene system based on the gene encoding Escherichia coli polyphosphate kinase (PPK), which can be used to monitor gene expression in mammalian cells. PPK catalyzes the synthesis of inorganic polyphosphate (polyP) from ATP, and because mammalian cells do not contain detectable levels of polyP, PPK activity can be measured in mammalian cells using 31P-magnetic resonance spectroscopy or 31P-magnetic resonance imaging. The ppk reporter gene system described here is noninvasive, does not require an exogenous substrate, and can potentially be used in internal tissues of living organisms.     

Tetracycline-regulated gene expression mediated by a novel chimeric repressor that recruits histone deacetylases in mammalian cells

Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be developed. Histone deacetylases have recently been shown to play an important role in regulating gene expression. Here we investigated whether a more tightly controlled expression could be achieved by using a novel chimeric repressor that recruits histone deacetylases to a tetracycline-responsive promoter. This chimeric repressor was engineered by fusing the tetracycline repressor (TetR) with an mSin3-interacting domain of human Mad1 and was shown to bind the tetO(2) element with high affinity, and its binding was efficiently abrogated by doxycycline. The chimeric repressor was shown to directly interact with mSin3 of the histone deacetylase complex. This inducible system was further simplified by using a single vector that contained both a chimeric repressor expression cassette and a tetracycline-responsive promoter. When transiently introduced into mammalian cells, the chimeric repressor system exhibited a significantly lower basal level of luciferase activity (up to 25-fold) than that of the TetR control. When stably transfected into HEK 293 cells, the chimeric repressor system was shown to exert a tight control of green fluorescent protein expression in a doxycycline dose- and time-dependent fashion. Therefore, this novel chimeric repressor provides an effective means for more tightly regulated gene expression, and the simplified inducible system may be used for a broad range of basic and clinical studies.     

Expression and processing of a bacterial endoglucanase in transgenic mice.

The C6.5 endoglucanase from Bacillus subtilis catalyzes the hydrolyses of beta-glucans. This enzyme, which is also produced by many ruminant microbes, is not part of the normal digestive repertoire of monogastric animals. We have generated transgenic mice which express the C6.5 endoglucanase gene specifically in the pancreas with secretion of the enzyme into the small intestine. The secreted enzyme has a molecular mass of 55 kDa which is reduced by protease digestion to the principal forms of 37 and 35 kDa. These truncated forms are resistant to further protease degradation and exhibit enhanced specific activity compared to the native enzyme. These results encourage further investigation of the utility of this transgene for enhancing the digestive capability of monogastric animals.     

Targeting of tumor cells by lymphocytes engineered to express chimeric receptor genes

Adoptive cellular immunotherapy of cancer has been limited to date mostly due to the poor immunogenicity of tumor cells, the immunocompromised status of cancer patients in advanced stages of their disease, and difficulties in raising sufficient numbers of autologous tumor-specific T lymphocytes. On the other hand, the slow tumor penetration and short half-life of exogenously administered tumor-specific monoclonal antibodies have provided major obstacles for an effective destruction of tumor cells by the humoral effector arm of the immune system. Attempts to improve the efficacy of adoptive cellular cancer immunotherapy have led to the development of novel strategies that combine advantages of T cell-based (i.e., efficient tumor penetration, cytokine release and cytotoxicity) and antibody-based (high specificity for tumor-associated antigens) immunotherapy by grafting cytotoxic T lymphocytes (CTLs) with chimeric receptors composed of antibody fragments (which recognize tumor-cell antigens) and a cellular activation motif. Antigen recognition is therefore not restricted by major histocompatibility genes, as the physiological T-cell receptor, but rather is directed to native cell surface structures. Since the requirements of major histocompatibility complex (MHC) restriction in the interaction of effector cells with target cells are bypassed, the tumor cell-binding of CTLs grafted with chimeric receptors is not affected by down-regulation of HLA class I antigens and by defects in the antigen-processing machinery. Ligand binding by the chimeric receptor triggers phosphorylation of immunoglobulin tyrosine activation motifs (ITAMs) in the cytoplasmic region of the molecule and this activates a signaling cascade that is required for the induction of cytotoxicity, cytokine secretion and proliferation. Here, the authors discuss the potential of lymphocytes grafted with chimeric antigen receptors in the immunotherapy of malignant disease.     

Efficient transformation of mammalian cells with constructs containing a puromycin-resistance marker

Recombinant plasmids have been obtained that lead to the accumulation of five- to ten-fold more puromycin-N-acetyl-transferase (PAC) mRNA and two- to three-fold more PAC activity than the already described plasmid pSV2pac [Vara et al., Nucl. Acids Res. 14 (1986) 4117-4124]. When these optimized recombinants were used for stable transformation to puromycin resistance, efficiencies up to 1 x 10(-2) were obtained, indicating that these pac-containing recombinants may be very useful dominant selectable markers for gene transfer in mammalian cells.     

Efficient transfer of chromosome-based DNA constructs into mammalian cells

Artificial chromosomes, engineered minichromosomes and other chromosome-based DNA constructs are promising new vectors for use in gene therapy, protein production and transgenics. However, a major drawback in the application of chromosome-based DNA is the lack of a suitable and convenient procedure for large-scale cellular introduction, which is particularly frustrated by their size (1 by 2 microm). Here we present a method to transfer Artificial Chromosome Expression systems (ACEs) into mammalian cells, which relies on a combined approach of using cationic amphiphiles and high frequency ultrasound. Thus, when cells were preincubated with liposomes consisting of the cationic lipid SAINT-2 and the phospholipid dioleoylphosphatidylethanolamine (molar ratio 1:1), followed by ultrasound, ACEs could be introduced into mammalian cells, which resulted in the expression of ACEs-harbored reporter genes, such as Green Fluorescent Protein. Depending on cell type, transfection efficiencies ranged from 12% to 53%. Interestingly, no detectable delivery occurred when cells were treated alone with either ultrasound or liposomes. Evidence is provided, based on cellular entry of differently sized beads and trypan-blue permeation, which supports a mechanism in which integration of the lipids creates unstable membrane domains, which are particularly prone to ultrasound-induced pore formation. Time- and temperature-dependent experiments indicate that these pores display a transient stability. Hence, following ultrasound, the pores disappear as a function of time as suggested by a time-window for ACEs entry, and trypan blue exclusion, 80% of the cells becoming stained immediately following ultrasound, dropping to approximately 20% after 30 min. Co-expression of different genes in conjunction with fluorescence in situ hybridization (FISH) analysis indicates that the current procedure provides a means to introduce functionally active artificial chromosomes into eukaryotic cells.     

Retrovirus-mediated gene expression in mammalian cells.

Significant advances have been made in precisely defining the elements in the Moloney murine leukemia virus genome responsible for tissue-restricted expression. This knowledge should lead to improved expression vectors for gene transfer in mammalian cells. In the past year, retrovirus-mediated gene expression in a diverse range of cell types has been reported. These cells have been used to study gene transfer relevant to a range of inherited diseases.     

Expression of a X. laevis tRNATyr gene in mammalian cells.

Expression of a X. laevis tRNATyr gene has been studied in mammalian cells. This tRNATyr gene has a 13 base intervening sequence adjacent to its anticodon. A fragment containing the tRNATyr gene was cloned into the late region of SV40. Cells infected with a recombinant virus stock vastly overproduce a tRNATyr that is properly spliced, processed and modified. It was also found that the X. laevis tRNATyr is identical or nearly identical to an endogenous tRNATyr of monkey kidney cells. The possibility of using the X. laevis tRNATyr gene to create an amber suppressor for mammalian cells is discussed.     

Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs

We evaluate here the use of real-time quantitative PCR (q-PCR) as a method for screening for homologous recombinants generated in mammalian cells from either conventional gene-targeting constructs or whole BAC-based constructs. Using gene-targeted events at different loci, we show that q-PCR is a highly sensitive and accurate method for screening for conventional gene targeting that can reduce the number of clones requiring follow-up screening by Southern blotting. We further compared q-PCR to fluorescent in situ hybridization (FISH) for the detection of gene-targeting events using full-length BAC-based constructs designed to introduce mutations either into one gene or simultaneously into two adjacent genes. We find that although BAC-based constructs appeared to have high rates of homologous recombination when evaluated by FISH, screening by FISH was prone to false positives that were detected by q-PCR. Our results demonstrate the utility of q-PCR as a screening tool for gene targeting and further highlight potential problems with the use of whole BAC-based constructs for homologous recombination.