Bacterial Neomycin Resistance Research Articles

An antibiotic selection marker for nematode transgenesis

We have developed a nematode transformation vector carrying the bacterial neomycin resistance gene (NeoR) and shown that it could confer resistance to G-418 on both wild-type Caenorhabditis elegans and C. briggsae. This selection system allows hands-off maintenance and enrichment of transgenic worms carrying non-integrated transgenes on selective plates. We also show that this marker can be used for Mos1-mediated single-copy insertion in wild-type genetic backgrounds (MosSCI-biotic).

A targeted deletion/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus ( Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene ( neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1 +/− mice produced no Pcsk1 −/− offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1 +/− . These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT–PCR analysis on RNA from endocrine tissues from Pcsk1 +/− mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1 +/− and Pcsk1 +/+ mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele.

Relative rates of insertion and deletion mutations in dinucleotide repeats of various lengths in mismatch repair proficient mouse and mismatch repair deficient human cells

Microsatellites are DNA elements composed of short tandem repeats of 1–5 bp. These sequences are particularly prone to frameshift mutation by insertion–deletion loop formation during replication. The mismatch repair system is responsible for correcting these replication errors, and microsatellite mutation rates are significantly elevated in the absence of mismatch repair. We have investigated the effect of varying the number of repeats in a (CA) n microsatellite on mutation rates in cultured mammalian cells proficient or deficient in mismatch repair. We have also compared the relative rates of single-repeat insertions and deletions in these cells. Two plasmid vectors were constructed for each repeat unit number ( n=8, 17, and 30), such that the microsatellites, placed upstream of a bacterial neomycin resistance gene ( neo), disrupted the reading frame of the gene in the (−1) or (+1) direction. Plasmids were introduced separately into the cells, where they integrated into the cellular genome. Mutation rates were determined by selection of clones with frameshift mutations in the microsatellite that restored the reading frame of the neo gene. We found that mutation rates were significantly higher for (CA) 17 and (CA) 30 tracts than for (CA) 8 tracts in both mismatch repair proficient (mouse) and deficient (human) cells. A mutational bias favoring insertions was generally observed. In both (CA) 17 and (CA) 30 tracts, single-repeat insertion rates were higher than single-repeat deletion rates with or without mismatch repair; deletions of multiple repeat units (≥8 bp) were observed in these tracts, where as deletions this large were not found in the (CA) 8 tract. Single-repeat mutations of both types were made at similar rates in (CA) 8 tracts in human mismatch repair deficient (MMR −) cells, but single-repeat insertion rates were higher than single-repeat deletion rates in mouse mismatch repair proficient (MMR +) cells. Results of these direct studies on microsatellite mutations in cultured cells should be useful for refinement of mathematical models for microsatellite evolution.

Relative stabilities of dinucleotide and tetranucleotide repeats in cultured mammalian cells.

The differences in rates of frameshift mutations between a dinucleotide repeat sequence [(CA)(17)] and a tetranucleotide repeat sequence [(GAAA)(17)] have been determined in immortalized, non-tumorigenic, mismatch repair-proficient mouse cells and in mismatch repair-defective human colorectal cancer cells. Clones with mutations were selected on the basis of restoration of activity of a bacterial neomycin resistance gene whose reading frame was disrupted by insertion of the microsatellite upstream of the translation initiation codon. This gene was introduced into the cells on a plasmid, which integrated into the genome of the host cells. Mutation rates of the tetra-nucleotide repeat were much lower than those of the dinucleotide repeat in both cell types. In addition, independent subclones of the colorectal cancer cell line were assayed by PCR for instability of endo-gen-ous tetranucleotide and dinucleotide repeat sequen-ces. In all cases, the mutation frequencies of the dinucleotide repeats were higher than those of the tetranucleotide repeats.

Characterization of adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice

Using transgenic mice, we targeted SV40 T antigen and the bacterial neomycin resistance gene to steroidogenic tissues using a human P450 cholesterol side-chain cleavage promoter. Expression of SV40 T antigen resulted in adrenocortical tumors. Adrenocortical cell lines from one of these tumors (ST5R) was previously characterized. We have now obtained clonal lines from the second more differentiated tumor. After dispersion of the left adrenal tumor, ST5L parental cells were selected with G418 and subcloned. The resulting adrenocortical subcloned cell lines are more highly differentiated than those cell lines resulting from the right adrenal tumor (ST5R). ST5L cell lines secrete progesterone and corticosterone to varying degrees, whereas ST5R cells secrete only progesterone. One of the clonal cell lines, ST5Lc16, expresses both P450c11β and P450c11AS mRNAs, which normally are regionally distributed in different zones of the adrenal cortex. Thus, ST5Lc16 cells may be progenitor cells for both glomerulosa and fasciculata cells and may provide clues to the cellular and molecular events leading to the differentiation of the glomerulosa and the fasciculata-reticularis. Other ST5Lc cell lines are more representative of the fasciculata-reticularis, because they express P450c11β mRNA and secrete corticosterone, and they neither express P450c11AS mRNA nor do they secrete aldosterone. All cell lines also have 21-hydroxylase activity, but none express P450c21, indicating that some other, as yet unidentified, enzyme has this activity. In all cell lines, steroid secretion is regulable by cAMP stimulation but not by ACTH stimulation. All ST5L cell lines also express mouse renin-1 mRNA. In addition to their utility in studies of adrenal steroidogenesis, these cell lines may also be useful in studying the etiology of adrenocortical tumors.

The Developmental and Physiological Consequences of Disrupting Genes Encoding β1 and β2 Adrenoceptors

Publisher Summary The role that individual β AR subtypes play in specific physiological processes has been traditionally defined using subtype-specific ligands. Unfortunately, many of these ligands either possess poor selectivity or are used at concentrations in vivo that can lead to occupation of nonspecific subtypes. The advent of gene disruption techniques in the mouse now enables to selectively delete or alter cloned genes as a means of identifying the specific function(s) of their gene products. To better understand β AR subtype-specific functions in the context of either the whole animal or isolated organs and cells, the genes encoding both β 1 - and β 2 ARs have been disrupted. Gene targeting vectors containing β 1 AR or β 2 AR gene sequences interrupted or partially replaced by a bacterial neomycin resistance gene cassette were flanked by a viral thymidine kinase gene cassette. A standard positive-negative selection strategy (G418 + gancyclovir) has been used to isolate R1 embryonic stem (ES) cells, having undergone homologous recombination at β 1 AR or β 2 AR loci. The pharmacological profile of β 1 AR and β 2 AR knockouts is consistent with the loss of these specific receptor subtypes. When the nonspecific β AR antagonist [ 125 I]cyanopindolol is used in competition binding studies, excess unlabeled CGP 20712A ( β 1 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 1 AR knockout heart or lung membranes while excess unlabeled ICI 118,551 ( β 2 AR-specific antagonist) reveals a selective loss of specific high-affinity sites in β 2 AR knockout lung membranes. These results provide further evidence that the β 1 AR and β 2 AR gene disruptions have abolished β AR protein expression. The physiological impact of knocking out either β 1 - or β 2 ARs has been studied in instrumented animals and in isolated tissues.

A Bicistronic Retrovirus Vector Containing a Picornavirus Internal Ribosome Entry Site Allows for Correction of X-Linked CGD by Selection for MDR1 Expression

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

Expression of VL30 Vectors in Human Cells That Are Targets for Gene Therapy

Novel vectors made from mouse VL30 retrotransposons were tested in cell types that are targets for gene therapy, including normal human cells (skeletal muscle epithelium, bronchial epithelium, mammary epithelium), Epstein-Barr virus-transformed peripheral blood lymphocytes (PBLs), and various tumor cell lines. The long terminal repeat (LTR) transcriptional promoter, derived from the retroelement NVL-3, expressed abundant mRNA containing the bacterial neomycin resistance gene (neo) in all cell types tested. The amounts of neo RNA detected on RNA blots from normal vs. transformed cells were similar, although relatively less RNA was expressed in PBLs than in other cell types. Vector RNA expression in PBLs persisted during six months of continuous culture. Transcription was regulated by fibroblast growth factor (bFGF) and insulin, with the effects of each being cell-type-dependent. Thus, VL30 vectors introduced and expressed transgenes at significant levels in a number of cell types that are of interest for human gene therapy of metabolic and neoplastic diseases.

Stable expression and functional characterization of a human cardiac Na + channel gene in mammalian cells

D. S. Krafte, W. A. Volberg, L. Rapp, R. G. Kallen, P. H. Lalik and R. B. Ciccarelli. Stable Expression and Functional Characterization of a Human Cardiac Na + Channel Gene in Mammalian Cells. Journal of Molecular and Cellular Cardiology (1995) 27, 823–830. In order to develop mammalian cell lines expressing a functional human heart Na + channel gene (hH1), Chinese hamster ovary (CHO-K1) cells and HeLa cells were transfected with the hill gene and the bacterial neomycin (neo) resistance gene. In CHO-K1 cells, direct screening for hH1-positive, G418-resistant colonies by functional patch clamp analysis was complicated due to low-level endogenous expression of a brain-type Na + channel. Therefore, we developed a stepwise strategy for isolation of cell lines expressing functional hH1 Na + channels: G418-resistant colonies were sequentially analysed for (1) chromosomal integration of hill DNA by PCR, (2) specific hill rnRNA expression by RT-PCR, (3) hH1 protein production by immunoprecipitation with hH1-specific antisera, and (4) hH1 Na + channel function by patch-clamp analysis. Using this strategy we obtained two CHO-K1 cell lines which express functional human heart Na + channels. However, using the same strategy, we were unsuccessful in obtaining functional, hH1-positive HeLa cell lines, even though hill mRNA and protein was produced in these ceils. The two CHO-K1 cell lines stably express human cardiac Na + channels which retain normal electrophysiological characteristics with respect to activation and inactivation. In addition, the Na + channels expressed in these cells are blocked by tetrodotoxin with an IC 50 value of 2.5 μ m; consistent with known cardiac Na + channel pharmacology. The density of channels is high enough to permit recording of pseudomacroscopic currents in excised outside-out patches of membrane. Stable expression of the human heart Na + channel gene in non-cardiac mammalian cells further indicates many of the distinguishing properties of these channels are encoded by this gene. In addition, the CHO-K1 cell system should prove useful in the further molecular, biochemical and biophysical characterization of human cardiac Na + channels.

An efficient expression vector for stable expression in human liver cells

The expression of the bacterial neomycin resistance (Nm R)-encoding gene was examined under control of the promoter region of the gene encoding the human polypeptide chain elongation factor lot (EF-loc). In a human liver cell line, HepG2, the plasmid pEF321- neo directed higher expression of the bacterial neo gene than the Nm R gene expression vectors using other promoters, like the SV40 early region or thymidine kinase of Herpes simplex virus and SV40 early region promoter linked to human T-cell leukemia virus-1 enhancer sequences.

Inhibition of Differentiation in P19 Embryonal Carcinoma Cells by the Expression of Vectors Encoding Truncated or Antisense EGF Receptor

Murine embryonal carcinoma cells do not express detectable cell-surface epidermal growth factor receptors (EGF-R) but after 2 days of differentiation induced by retinoic acid (RA) increasingly express mRNA and protein encoded by the EGF receptor gene (Joh et al., Cell Growth and Differentiation 3, 315, 1992). The effect on morphology, growth, and differentiation of the introduction of expression vectors that produce either a truncated, kinase-negative mouse EGF receptor or an antisense mRNA was studied in P19 embryonal carcinoma (EC) cells before and after differentiation. The presence of either construct should lead to the reduction of EGF-R expression by either the dominant negative effects of a truncated protein or the inhibition of endogenous EGF-R mRNA production/translation by complementary RNA, respectively. Cells were cotransfected with the bacterial neomycin resistance gene and constitutively expressing clones were selected with G418. The cytomegalovirus LTR promoter/enhancer was found to be very inefficiently activated in Pl9 EC cells. After RA addition, changes in gene expression included induction of both the exogenous truncated constructs and endogenous EGF-R. Differentiation was gauged by the expression of tissue-type plasminogen activator and intermediate filament protein markers of neural tissues, as well as EGF-R. The expression of 120-kDa truncated EGF-Rs was high in four clones, but all 10 clones examined had diminished abilities to differentiate after RA induction compared to four control cell lines. Similarly, the majority of antisense transfected clones was unable to differentiate normally. The results indicate that the reduced expression of EGF-Rs in differentiating EC cells inhibits the rate, frequency, and extent of differentiation after RA induction. We conclude that the expression of EGF-Rs plays a role in the stimulation of differentiation and we speculate that the mechanism involves the tyrosine kinase activity of the receptor.

Regulation of the cellular thymidine kinase gene promoter in simian virus 40-infected cells

We examined the regulation of the cellular thymidine kinase (TK) gene promoter in simian virus 40 (SV40)-infected simian CV1 cells. Nuclear run-on transcription assays demonstrated a three- to fourfold increase in the rate of transcription of the endogenous gene at 14 to 16 h following viral infection. In addition, hybrid genes containing the human TK promoter linked to the bacterial neomycin resistance gene were induced by SV40 in stably transfected cells, indicating that promoter sequences are sufficient to confer viral regulation. Analysis of human TK promoter deletion mutants indicated that sequences localized between -67 and +30 bp relative to the transcriptional initiation site are sufficient to confer regulation on SV40-infected cells. These sequence elements are distinct from those required for serum induction, which were previously localized to the region between -135 and -67. These results suggest that SV40 activates novel cellular pathways that are not activated by serum stimulation of quiescent cells.

A plasmid system to monitor gene conversion and reciprocal recombination in vitro

A plasmid system allowing for the detection of recombinagenic activitues in cell-free extracts is described. Two truncated alleles of the bacterial neomycin resistance gene ( neo), differing from each other at a polymorphic restriction site, were constructed. Recombinations involving both alleles mediated by Drosophila embryo nuclear protein extracts or Drosophila larva whole cell protein extracts were selected by their ability to confer kanamycin resistance to E. coli. Restriction analysis of plasmids recovered from E. coli transformants allowed the monitoring of the two molecular mechanisms which can lead to functional neo genes, gene conversion and reciprocal recombination. A dose dependent increase in the recombination frequency with increasing amounts of cell extract was observed. Recombination was further increased by linearizing one of the two substrate plasmids. The Drosophila cell extracts catalyzed recombination in vitro since after incubation a recombination product could be identified by polymerase chain reaction (PCR) technology. The recombination was absolutely dependent on the presence of an active cell extract, since no diagnostic PCR product was detected in a reaction where extract was omitted. Analysis of a representative number of recombinant plasmids by restriction analysis revealed that in the absence of an exogenous recombinational system less than 2% of kanamycin resistant recombinant plasmids occurred by gene conversion upon transformation into E. coli. In contrast, recombinants exhibiting restriction patterns diagnostic for gene conversion were observed at frequencies between 5.1% and 9.8% after incubation with Drosophila larva cell extracts. These results strongly argued that gene conversion is a prominent mechanism of recombination in Drosophila mitotic cells.

The 35-kilodalton protein gene (p35) of Autographa californica nuclear polyhedrosis virus and the neomycin resistance gene provide dominant selection of recombinant baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) recombinants were constructed to test the effectiveness of the AcMNPV 35-kilodalton protein gene (35K gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. Insertion of the AcMNPV apoptosis suppressor gene (p35) into the genome of p35-deletion mutants inhibited premature host cell death and increased virus yields up to 1200-fold at low multiplicities in Spodoptera frugiperda (SF21) cell cultures. When placed under control of an early virus promoter, the bacterial neomycin resistance gene (neo) restored multiplication of AcMNPV in the same cells treated with concentrations of the antibiotic G418 that inhibited wild-type virus growth greater than 1000-fold. The selectivity of these dominant markers was compared by serial passage of recombinant virus mixtures. After four passages, the proportion of p35-containing virus increased as much as 2,000,000-fold relative to deletion mutants, whereas the proportion of neo-containing viruses increased 500-fold relative to wild-type virus under G418 selection. The strength and utility of p35 as a selectable marker was further demonstrated by the construction of AcMNPV expression vectors using polyhedrin-based transfer plasmids that contain p35. Recombinant viruses with foreign gene insertions at the polyhedrin locus accounted for 15 to 30% of the transfection progeny. The proportion of desired viruses was increased to greater than 90% by linearizing the parental virus DNA at the intended site of recombination prior to transfection. These results indicate that p35 and neo facilitate the selection of baculovirus recombinants and that p35, in particular, is an effective marker for the generation of AcMNPV expression vectors.

Molecular and functional characterization of the murine glucocerebrosidase gene

A genomic clone of glucocerebrosidase (D-glucosyl-N-acyl-sphingosine glucohydrolase; E.C. 3.2.1.45) purified from a genomic library derived from a Balb/c mouse was analyzed by restriction mapping and nucleotide sequencing of its promoter and protein coding regions. Promoter activity was functionally assessed by ligation of a 2 kb glucocerebrosidase fragment to the protein coding segment of a bacterial neomycin resistance gene. Smaller segments of the 5' flanking sequence were then analyzed for their ability to initiate transcription of the chloramphenicol acetyltransferase reporter gene. A 319 bp Eco RI-Bgl II fragment (containing 259 bp upstream of the cDNA 5' limit) ligated to the chloramphenicol acetyltransferase open reading frame produced considerable activity.

Establishment of an osteogenic cell line derived from adult mouse bone marrow stroma by use of a recombinant retrovirus.

In order to characterize fibroblastic colony-forming units (CFU-F) from murine bone marrow in relation to osteogenesis, adherent cells of 7-day-old BALB/c mouse bone marrow cultures were infected with a recombinant retrovirus (N2/ delta fosB) containing the bacterial neomycin resistance gene. One of the G418-resistant clones, MN7, was selected for further analysis on the basis of its high expression of the bone-specific alkaline phosphatase. The cells have now been in culture for more than 1 year and maintain a stable phenotype. The osteogenic nature of the immortalized clone MN7 was demonstrated as follows: (1) Mineralization was detected by 85Sr uptake and with the Von Kossa staining method only after in vitro cultivation on a collagen type I matrix. (2) Osteoblastic phenotype markers, including the synthesis of type I collagen, osteonectin, and the bone-specific isoenzyme of alkaline phosphatase were expressed in vitro. (3) MN7 cells responded to bone effectors such as parathyroid hormone and 1,25-dihydroxyvitamin D3. (4) Intraperitoneal injection of MN7 cells into 1-day-old BALB/c mice produced typical osteosarcomas in all animals. We conclude that MN7, derived entirely in vitro from a stromal CFU-F colony, represents a stable murine osteosarcoma cell line expressing the osteoblastic phenotype and provides the first direct evidence needed to establish adult mouse marrow-derived, nonhematopoietic stromal cells as osteoprogenitors.

Retrotransposon gene engineering.

We have used a mobile mouse VL30 genetic element together with retroviral helper cells to efficiently transmit and express chimeric foreign gene sequences in murine and human cells. The construct comprised a cDNA copy of retrotransposon NVL3, an internal promoter [rat cytosolic phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32)] and an expressed bacterial neomycin resistance gene. Thirty to sixty thousand colony forming units/ml (CFU/ml) were recovered from the supernatant of mass cultured psi2 helper cells transfected with the recombinant retrotransposon plasmid DNA. RNA was expressed from both the VL30 long terminal repeat and from the internal PEPCK promoter, resulting in a G418 drug resistance phenotype in recipient cells. Integrated VL30 DNA sequences transduced from psi2 or PA317 retroviral helper cells failed to regenerate detectable replication competent virus. Human and rodent recipient cells transduced by the retrotransposons appeared to bear intact vector sequences after two rounds of transmission by helper cells.

Expression of a Foreign Gene in Cats Reconstituted With Retroviral Vector Infected Autologous Bone Marrow

A Moloney murine leukemia virus based retroviral vector was used to transfer the bacterial neomycin resistance gene (neoR) into feline hematopoietic cells. We reconstituted four cats that had been lethally irradiated with autologous bone marrow that had been infected with the N2 or SAX retroviral vector. Bone marrow cells from all four cats expressed the neoR gene 30 days posttransplant and three of four cats still had the neoR gene and a low level of drug resistant colony-forming unit granulocyte-macrophage after more than 200 days. Two of the four cats unexpectedly developed diabetes mellitus 90 days posttransplantation. The expression of a foreign gene in cats, albeit at a low level, demonstrates that retroviral vectors can be used for gene transfer in noninbred large animal species and may be useful for gene therapy of humans. The development of diabetes mellitus in two of the subjects emphasizes the value of animal models for the study of possible deleterious effects of retroviral vector-mediated gene transfer.

Expression of a foreign gene in cats reconstituted with retroviral vector infected autologous bone marrow

A Moloney murine leukemia virus based retroviral vector was used to transfer the bacterial neomycin resistance gene (neoR) into feline hematopoietic cells. We reconstituted four cats that had been lethally irradiated with autologous bone marrow that had been infected with the N2 or SAX retroviral vector. Bone marrow cells from all four cats expressed the neoR gene 30 days posttransplant and three of four cats still had the neoR gene and a low level of drug resistant colony- forming unit granulocyte-macrophage after more than 200 days. Two of the four cats unexpectedly developed diabetes mellitus 90 days posttransplantation. The expression of a foreign gene in cats, albeit at a low level, demonstrates that retroviral vectors can be used for gene transfer in noninbred large animal species and may be useful for gene therapy of humans. The development of diabetes mellitus in two of the subjects emphasizes the value of animal models for the study of possible deleterious effects of retroviral vector-mediated gene transfer.