A rapid procedure for screening fibroblast packaging cell lines for secretion of selectable retrovirus

A simple method is described for rapidly screening fibroblast packaging cell line clones for high-titer secretion of retroviruses carrying a selectable marker. Virus-containing supernatants are used to infect FDC-P1 myeloid cells in 24-well tissue culture plates, selection is applied and wells containing live cells (infected by retrovirus and thus expressing the selectable marker) are detected after 5 to 10 days. The number of live cells in each well is proportional to the retroviral titer of the infecting supernatant. The assay is quick to set up and allows simultaneous screening of many samples.     

Analysis of FHIT transcripts in cervical and endometrial cancers

We show that mammalian cells can be stably transfected by a mechanical loading procedure in which cells are forced through a small opening in the presence of DNA. A suspension of cells and plasmid DNA in growth medium was passed up and down through a 30-gauge needle attached to a 1-ml syringe. Cells were immediately plated at appropriate densities for subsequent selection for stable expression of a marker gene. Two rodent cell lines, Chinese hamster ovary and mouse Ltk- cells, were successfully transfected with an efficiency of about one transfectant per 5 x 10(4) cells. The human HeLa cell line was transfected with a somewhat lower efficiency. Pluronic F-68, a detergent believed to aid in healing of membrane injuries, had no beneficial effect when present during the loading procedure. Successful transfection was accomplished using three different genes as selectable markers. Southern blotting analysis revealed that transfectants contained one or very few copies of the introduced DNA construct integrated into the genome. Several transfectants were demonstrated to remain stable for more than 20 generations of growth in the absence of selection. This procedure is fast, economical, and of general utility.     

Development of a new easy complementation assay for DNA repair deficient human syndromes using cloned repair genes

Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.     

The production of a stably transformed insect cell line expressing an invertebrate GABAA receptor beta-subunit

We have produced a stable insect cell line derived from Spodoptera frugiperda (Sf9) cells expressing a cDNA encoding a beta-subunit of the Lymnaea stagnalis GABAA receptor. The cDNA was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (IE-1) promoter. Stable cell lines were established by transformation of Sf9 cells with the expression vector pIEK1. LGbeta1 together with a plasmid encoding a selectable marker which confers neomycin (G418) resistance. Following growth in the presence of G418, neomycin resistant clones were selected, amplified and analysed for the presence of functional GABA-gated chloride channels. Electrophysiological analysis of one cell line showed the presence of a picrotoxin-sensitive chloride channel not present in control Sf9 cells. These channels were also sensitive to GABA, albeit at relatively high (mM) concentrations.     

Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene

A cyclic AMP-responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose-dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein-coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5-HT) receptor agonist ligands caused an inhibition of forskolin-stimulated luciferase expression in the rank order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin. The response to 5-HT was reversed by the 5-HT1 receptor antagonists cyanopindolol and pindolol, but not the 5-HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene-related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8-37). These results were consistent with the presence of 5-HT(1B-like) and calcitonin (C1a-like) receptors in CHO cells, with the responses to 5-HT and CGRP being pertussis and cholera toxin-sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein-coupled receptors linked to adenylyl cyclase.     

In vitro lipid peroxidation of LDL from postmenopausal cynomolgus macaques treated with female hormones

We constructed a sensitive and quantitative assay system to examine human T-cell leukemia virus type I (HTLV-I) envelope (env) glycoprotein-mediated cell fusion in which T7 RNA polymerase in donor cells coexpressing env glycoproteins activates a reporter gene in recipient cells upon cell fusion. An efficient expression of HTLV-I env glycoproteins (gp46 and gp21) was observed in 293T cells transfected with an expression plasmid by both immunoblot and immunofluorescence analyses. The cells expressing env glycoproteins also exhibited self-fusion. By cocultivating the donor cells with recipient cells transfected with a reporter plasmid possessing the luciferase gene under the T7 promoter, the expression of luciferase was observed upon cell fusion. The activation of the luciferase gene was inhibited by either anti-env neutralizing antibody or synthetic peptide corresponding to env gp21, thus indicating the cell fusion to be specifically mediated by the HTLV-I env glycoproteins expressed in the donor cells. A broad range of cell lines exhibited susceptibility to HTLV-I env-mediated cell fusion by this assay. This newly established assay system may thus provide an efficient way both to study the fusion mechanisms mediated by HTLV-I env glycoproteins and to identify the HTLV-I receptor(s).     

Transgenic mice for the establishment of histidinol-resistant embryonic fibroblast feeder layers

Gene targeting in mouse embryonic stem cells generates mutations by replacing an endogenous chromosomal region with a copy disrupted by a selectable genetic marker. The most commonly used selectable marker is the bacterial neo(r) gene, which confers resistance in mammalian cells to the antibiotic G418. Use of an alternative selectable marker, the Salmonella typhimurium gene hisD, should provide expanded applications for gene targeting. The hisD gene encodes the protein histidinol dehydrogenase, which catalyzes the conversion of histidinol to the amino acid histidine. Histidinol is toxic to mammalian cells, while histidine is an essential mammalian amino acid. Consequently, growth selection in cultures with media containing histidinol in place of histidine occurs by both histidine starvation and histidinol poisoning. The hisD selection is being tested for potential use in gene targeting experiments with mouse embryonic stem (ES) cells. Currently, most successful gene targeting experiments use primary embryonic fibroblast feeder layers, which assist in the maintenance of the pluripotential state of the embryonic stem cells. To support ES cell stability under histidinol selection, mice transgenic for the S. typhimurium hisD gene have been produced and used to generate embryonic fibroblast feeder cells. The transgenic embryonic fibroblasts survive under a wide range of histidinol-containing growth conditions and support growth of ES cell cultures.     

Efficient long-term coexpression of a hammerhead ribozyme targeted to the U5 region of HIV-1 LTR by linkage to the multidrug-resistance gene

Ribozymes as anti-HIV-1 agents hold promise for the treatment of AIDS. They can be delivered into cells either exogenously or through an expression system. For effective protection against HIV-1, sufficient and sustained amounts of the antiviral ribozymes must be delivered into target cells. The coexpression of a dominant selectable marker with ribozymes would serve to enrich for cells containing the molecular antiviral and facilitate prolonged expression of these ribozymes. The multidrug resistance gene (MDR1) is a potential clinically relevant selectable marker and offers many advantages over other known dominant selectable markers, including the use of diverse pharmacologically characterized drug or drug combinations for selection. Harvey sarcoma-based retroviral vectors encoding the MDR1 multidrug transporter with a hammerhead ribozyme targeted to highly conserved sequences within the HIV-1 U5 LTR segment have been constructed in a bicistronic format. The internal ribosome entry site (IRES) from encephalomyocarditis virus was used to initiate translation of the MDR1 mRNA. The ribozyme remained functional despite being tethered to MDR1. Long-term, high-level expression of both the ribozyme and MDR1, as evident by RT-PCR and FACS analysis, was observed in a human T cell line containing the construct selected with vincristine, a cytotoxic substrate for the multidrug transporter.     

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

Concentration-dependent differential effects of N-acetyl-L-cysteine on the expression of HSP70 and metallothionein genes induced by cadmium in human amniotic cells

We have developed a stable DNA transfection vector pRANneo for genetic manipulation of the primitive protozoan Giardia lamblia. pRANneo was constructed by replacing the protein coding region of a Giardia ran gene with a bacterial neomycin phosphotransferase gene (neo). This plasmid was electroporated into G. lamblia, and the transfectants were selected by G418. pRANneo replicated episomally to approximately 80 copies per G. lamblia trophozoite as demonstrated by dot hybridizations, Southern hybridizations and transformations of the DpnI-treated plasmids into Escherichia coli. pRANneo/GDHluc was then constructed by incorporation of a luciferase expression system into pRANneo to persistently express firefly luciferase in G. lamblia under G418 selection. The NEO and luciferase proteins were detected in the transfected G. lamblia cells by Western blottings. The level of luciferase activity and the plasmid copy number correlated with the concentration of G418. Removal of G418 from the transfectant culture resulted in gradual loss of the plasmid and luciferase activity. The stable DNA transfection system should provide a valuable tool for genetic studies of G. lamblia.     

Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii

We have engineered a mutant version of the green fluorescent protein GFP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1996;173:33-38) for expression in the protozoan parasite Toxoplasma gondii. Although intact GFP was not expressed at any detectable level, GFP fusion proteins could be detected by fluorescence microscopy, flow cytometry (FACS), and immunoblotting. Both extracellular tachyzoites and T. gondii-infected host cells could readily be sorted by FACS, which should facilitate a variety of selection strategies. Several selectable markers were tested for their ability to produce stable green transgenic parasites. Fluorescence intensity was directly correlated with gene copy number and protein expression level. Weak selectable markers such as chloramphenicol acetyl transferase (CAT) driven by the SAG1 promoter, which yield multicopy insertions, are therefore most effective for selecting green fluorescent parasites-particularly when coupled to constructs which employ a strong promoter to drive GFP expression. Transformation vectors developed in the course of this work should be of general utility for the overexpression of heterologous transgenes in Toxoplasma. CAT-GFP fusion proteins were expressed in the parasite cytoplasm. GFP fusions to the P30 major surface antigen (linked on the same plasmid to a CAT selectable marker under control of various promoters) could be detected in dense granules within living cells, and were efficiently secreted into the parasitophorous vacuole. GFP fusions to the rhoptry protein ROP1 were targeted to rhoptries (specialized secretory organelles at the apical end of the parasite).     

ATP potentiates interleukin-1 beta-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.     

Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum

We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional beta-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.     

Crime scene and criminal findings site of an unknown cadaver in the field: reconstruction based on traces and clinical findings

The introduction of cloned DNA into mammalian cells allows functional testing of genes contained on the fragments. In many cases, the exogenous DNA introduced into mammalian cells requires selectable genes that mark the presence of input DNA. Two new vectors, carrying mammalian selectable markers encoding for either neomycin-resistance (neo) or histidinol-resistance (hol), have been constructed for targeted integration to specific single-copy sites within yeast artificial chromosome (YAC) insert DNA. The integration cassettes comprise a single selectable yeast gene adjacent to a mammalian selectable gene, either LEU2 with neo or HIS3 with hol. Modification of the YAC occurs in yeast by transfection with linear DNA containing YAC-specific, unique, recombinogenic ends, thereby ensuring co-integration of the markers. Analysis of modified YACs confirms that both vectors correctly integrate into the targeted unique sites. The precise localization of selectable marker genes in the cloned DNA ensures the integrity of the genomic fragments during functional testing. Placement of mammalian selectable markers within the YAC insert DNA should allow for YAC-based gene targeting experiments in a variety of mammalian cell lines.     

Mouse germinal center B cells with the xid mutation retain responsiveness to antimouse CD40 antibodies but diminish IL-5 responsiveness

The germinal center (GC) develops in secondary lymphoid tissues in response to thymus-dependent (TD) antigens. To investigate the molecular mechanism of B cell differentiation in GC, we enriched GC B cells from spleen of TD antigen-immunized wild-type and X-linked immunodeficient (XID) mice, and examined the differentiation of GC B cells into antigen-specific IgG1 antibody-forming cells (AFC) in response to anti-CD40 mAb and cytokines. A significant proportion of freshly purified GC B cells expressed receptors for IL-4 and IL-5. Anti-CD40 mAb sustained the viability of GC B cells and IL-4 co-operated with anti-CD40 mAb for further enhancement of the cell viability. Anti-CD40 mAb and IL-4 were essential for inducing differentiation of GC B cells into antigen-specific IgG1-AFC and IL-5 efficiently enhanced their differentiation. GC B cells with the xid mutation responded for proliferation to CD40 ligation to a lesser extent and for the IgG1-AFC response to anti-CD40 mAb together with IL-4, but they showed impaired responsiveness to IL-5, regardless of enhanced expression of IL-5R in response to anti-CD40 mAb and IL-4. These results suggest that anti-CD40 mAb, IL-4 and IL-5 play a critical role in the differentiation of mouse GC B cells. The GC B cells from XID mice show a functional defect with respect to IL-5-mediated differentiation.