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At least 307 records · Page 17Linked to original sources
PubMed1994

c-Ha-rasEJ transfection of rat aortic smooth muscle cells induces epidermal growth factor responsiveness and characteristics of a malignant phenotype.

Although the role of several protooncogenes, including sis, myc, and myb in the regulation of growth and differentiation of vascular cells has been examined in some detail, limited information is available on the contribution of ras genes to these processes. In the present studies the influence of oncogenic ras transfection on the phenotypic expression of rat aortic smooth muscle cells (SMCs) was examined. Cultured rat aortic SMCs during early passage (P4) were transfected by lipofection with c-Ha-rasEJ in a pSV2 neo vector or with pSV2 neo vector alone. Stable transfectants were selected in G418 over a 6-week period. Oncogene-transfected cells (ras-LF-1) exhibited differences in morphology and growth pattern relative to vector controls (neo-LF-1), or naive SMCs, including the development of prominent processes and the appearance of focal cellular arrangements giving rise to latticelike structures. Southern analysis revealed multiple integration of oncogenic ras in ras LF-1 cells. Transfection of c-Ha-rasEJ was associated with a twofold increase in p21 levels relative to pSV2 vector controls demonstrating that exogenous ras was expressed in these cells. Overexpression of ras p21 afforded SMCs a lower serum requirement for growth compared to vector controls, anchorage independent growth on soft agar, and acquisition of epidermal growth factor (EGF) responsiveness. Stimulation of serum-deprived SMCs with 5% fetal bovine serum (FBS) increased steady-state levels of c-Ha-ras mRNA in both ras-LF-1 and neo-LF-1 but ras induction was more pronounced in ras-transfected cells. alpha-smooth muscle (SM) actin gene expression was markedly reduced in ras-transfected cells relative to vector controls. These results show that transfection of c-Ha-rasEJ into aortic SMCs induces an altered phenotypic state characterized by alterations in growth factor-related signal transduction and tumorigenic potential.

Actins↗
PubMedDate not supplied

Transfection of epithelial cells is enhanced by combined treatment with mannitol and polyethyleneglycol.

BACKGROUND: Gene transfer efficiency drops significantly when polarized mammary epithelial cells are transfected instead of actively growing cells. However, fully differentiated cells are the targets for gene transfer in many in vivo applications. Therefore, a simple and effective method for the transfection of polarized mammary epithelial cells in confluent monolayers was developed. METHODS: Reporter gene plasmids were complexed with polyethylenimine with an average molecular weight of 25 kDa (PEI 25), or other agents, to transfect confluent monolayers of ovine mammary epithelial cells (OMEC II) or human carcinoma cells (CaCo-2) in vitro. The improved technique included pretreatment of the cells with a hyperosmotic mannitol solution (7%) which caused a loosening of the tight contacts between the cells. Alternatively, the mannitol shock could be replaced by a short treatment with trypsin or EDTA. In addition to the pretreatment, 12.5% polyethyleneglycol with an average molecular weight of 8000 kDa (PEG 8000) was included in the transfection mixture containing the DNA complexes. RESULTS: The combined application of mannitol and PEG resulted in a very reliable 5- to 30-fold increase in reporter gene expression in OMEC II and CaCo-2 cells, but not K562 cells (an example of another cell type). The improved technique can also be combined with other polymer-based transfection agents. The transfection rate was enhanced for confluent monolayer cells with fully developed epithelial polarity but also for subconfluent, growing epithelial cell cultures. CONCLUSIONS: A novel transfection protocol for epithelial cells is presented. The combined treatment of cells with mannitol and polyethyleneglycol results in substantial enhancement of in vitro transfection of epithelial cell lines.

Animals↗
PubMed2005

Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes.

BACKGROUND: One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application. METHODS: Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection efficiency were studied by luciferase assay and confocal microscopic observations. RESULTS: A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 microM GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency. CONCLUSION: The unique features of GALA to mediate improved transfection efficiencies were identified.

Animals↗
PubMed2006

Novel two-stage screening procedure leads to the identification of a new class of transfection enhancers.

BACKGROUND: Non-viral gene transfer efficiency is low as compared to viral vector systems. Here we describe the discovery of new drugs that are capable of enhancing non-viral gene transfer into mammalian cells using a novel two-stage screening procedure. METHODS: First, potential candidates are preselected from a molecular library at various concentrations by a semi-automated yeast transfection screen (YTS). The maximal transfection efficiency of every positive drug is subsequently determined in independent experiments at the optimal concentration and compared to the inhibitory effect of the drug on cell growth (IC50). In a subsequent mammalian cell transfection screen (MTS), the maximal transfection efficiency and the IC50 are determined for all preselected drugs using a human cell line and a luciferase reporter gene construct. RESULTS: Employing our novel system we have been able to identify a new class of transfection enhancers, the tricyclic antidepressants (i.e. doxepin, maprotiline, desipramine and amoxapine). All positive drugs enhanced gene transfer in both yeast and human cell lines, but lower concentrations were sufficient for mammalian cells. With a triple combination of doxepin, amoxapine and chloroquine we obtained a transfection efficiency that exceeded that of chloroquine, one of the best-known transfection enhancers of mammalian cells, by nearly one order of magnitude. CONCLUSIONS: Non-viral gene transfer efficiency can be increased significantly using new transfection enhancers that are identified by a novel, semi-automated two-stage screening system employing yeast cells in the first and specific human target cells in the second round.

Animals↗
PubMed1997

Relationship of ligand-receptor dynamics to actin polymerization in RBL-2H3 cells transfected with the human formyl peptide receptor.

The human formyl peptide receptor (FPR) expressed in RBL-2H3 transfectants (RBL[FPR]) behaves qualitatively like the FPR expressed by neutrophils except that it causes sustained F-actin accumulation and cell shape change responses on formyl peptide stimulation. These sustained responses were not accounted for by changes in the transfected receptor's ability to interact with ligand or by receptor density. Signal transduction pathways of transfected and neutrophil FPRs are apparently similar. In transfected cells, dissociation of ligand is sensitive to guanine nucleotide, the G protein is pertussis toxin-sensitive, FPR and G protein appear to be precoupled, the F-actin response is stimulated with the same dose-response profile as in neutrophils, and the F-actin accumulation response is directly regulated by the FPR, even long after initial stimulation. Potentially significant differences between neutrophil and transfected FPR were found when receptor processing was measured. In neutrophils, practically 100% of the FPR is converted to forms that dissociate slowly from ligand and are inactive in signal transduction within 2 min of ligand stimulation. By contrast, 20% or more of transfected FPR remains rapidly dissociating even 5 min after stimulation. Although 80% of neutrophil FPR is internalized by 5 min after stimulation, transfected FPR appears to plateau at 50-60% internalized. Because actin responses in neutrophils are regulated by a small number of active receptors, the inefficiency of receptor inactivation in RBL(FPR) transfectants may account for the prolonged F-actin accumulation response.

Actins↗
PubMed1992

Partial suppression of tumorigenicity in a human lung cancer cell line transfected with Krev-1.

A human non-small-cell lung carcinoma cell line, Calu-6 (from an anaplastic carcinoma), was transfected with the Ki-ras-related anti-oncogene Krev-1. Several transfectant lines were obtained that showed a reduced tumorigenicity in nude mice with respect to the parental and control transfected cell lines. This decrease was approximately 50% in tumor incidence at 4 wk after subcutaneous inoculation of the transfected cells. In addition, the volume of the Calu-6 revertant-derived tumors was three to 10 times smaller than that of the equivalent tumors produced by inoculation of the control cell line transfected with the neomycin-resistance gene. Krev-1--transfected cells that exhibited reduced tumorigenicity expressed Krev-1 mRNA and had variable numbers of copies of the Krev-1 gene. Moreover, Krev-1--transfected cells exhibited a more differentiated squamous epithelial morphology than the parental and control cell lines did. Moderately elevated levels of protein kinase C activity were detected in some revertant clones. Such activity correlated with the level of expression of Krev-1 mRNA in most cases. In summary, Krev-1 induced important morphological and biological changes in transfected Calu-6 cells that we interpreted as partial reversion of the malignant phenotype.

Animals↗
PubMed2004

Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons.

Numerous methods exist for transfecting postmitotic neurons, for example, DNA/calcium phosphate coprecipitation, cationic lipids, viruses, and physical methods such as microinjection, electroporation, and biolistics. Most methods, however, are either toxic to the cell, yield only poor transfection efficiencies, or cells have to be electroporated before plating. In this article, we present a standardized and fast transfection method using DNA/calcium phosphate coprecipitates that efficiently transfer DNA into mature, postmitotic hippocampal neurons. Shifting to CO(2)-independent media with a well-defined pH allows for the tight control of the coprecipitate formation and for adjusting the transfection parameters for the individual DNA plasmid used. The two critical parameters for reproducible and efficient transfections are: the precise pH during crystal formation, and the incubation time of the cells with the coprecipitate. This improved procedure now enables biochemical approaches. By transfecting a dominant-positive Ras mutant, we activate the Erk/MAP kinase signal transduction pathway. Furthermore, using a siRNA plasmid directed against MAP2, the level of an endogenously expressed protein is down-regulated upon transfection. These two approaches demonstrate that the presented transient transfection method can now be used to address questions on a biochemical level in hippocampal neurons.

Animals↗
PubMedDate not supplied

Efficient and reliable transfection of mouse erythroleukemia cells using cationic lipids.

Preliminary studies of cis-regulatory elements are frequently performed in transiently transfected cells before further analysis in stably transfected cell lines and transgenic mice. However, not all cells are readily transfectable by routine means. For instance, mouse erythroleukemia (MEL) cells have been a valuable model system for studies of their endogenous globin genes, but introduction of DNA using common transfection methods such as electroporation has been very inefficient. This has allowed studies of stably transfected cells, after selection for the rare transfection events, but transient transfection analysis has been problematic. This report describes an efficient and reliable method for transient transfection of MEL cells using commercially available cationic lipids.

Animals↗
PubMed1996

Inhibitory effect of double transfection to xenoendothelial cells using both decay accelerating factor and homologous restriction factor 20 genes on complement dependent cytolysis.

Hyperacute rejection in discordant xenotransplantation occurs due to the complement activation via classical and/or alternative pathway. Regulator of complement activation (RCA) molecules inhibit species-specific complement dependent cytolysis. To confirm the effect of gene engineering using double RCA molecules on xenogeneic cells, the inhibitory effect on complement dependent cytolysis was compared between bovine aortic endothelial cells (BAEC) doubly transfected with both decay accelerating factor (DAF) and homologous restriction factor (HRF) 20 cDNA and BAEC singly transfected with DAF alone using retroviral vector. The positive percent expression of DAF and HRF20 antigen on double transfectant (BAEC/D+H) was 97.0% and 95.0%, respectively, whereas that of DAF antigen on single transfectant (BAEC/D) was 97.0%. After incubated with 25% human serum and anti-BAEC antibody, the viability of double transfectant (BAEC/ D+H) was significantly preserved, compared with that of single transfectant (BAEC/D). These findings demonstrated that xenoendothelial cells doubly transfected with both DAF and HRF20 cDNA could be protected from complement dependent cytolysis more effectively than those singly transfected with DAF alone in the presence of antixenoendothelial antibody.

Animals↗
PubMed1989

Induction of urokinase activity and malignant phenotype in bladder carcinoma cells after transfection of the activated Ha-ras oncogene.

In order to characterize further the previously observed induction of a highly metastatic phenotype in mouse bladder carcinoma cells by Ha-ras transfection, we studied production of plasminogen activator, in vitro invasiveness, and the potential for lung colonization of these cells. The parent carcinoma cells produced predominantly tissue-type plasminogen activator. Out of 13 clones of ras-transfected cells tested, 8 secreted quantitatively elevated levels of plasminogen activator (up to 3.5-fold) as compared to the control transfectants. The plasminogen activator activity in cell lysates was maximally increased 3-fold, the surface-associated activity increased 2.5-fold. The secreted plasminogen activator of cloned ras-transfected cells was characterized to be predominantly of the urokinase type (71.3% compared to 20.5% with the parental BL cells). Thus, in addition to the quantitative augmentation of plasminogen activator production and secretion in a large fraction of the ras-transfected cell population, a significant qualitative shift from tissue-type to urokinase-type has been observed. In addition, ras-transfection augmented the capacity of the cells for invasion into Matrigel in a double-filter in vitro assay as well as their ability to colonize the lungs of syngeneic animals. These malignant properties of the transfected cells might be responsible for their highly metastatic behaviour induced by ras transfection.

Animals↗
PubMed1995

Efficient lipid-mediated transfection of DNA into primary rat hepatocytes.

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 micrograms of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 micrograms DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.

Animals↗
PubMed1997

Leukemia inhibitory factor (LIF) modulates pro-opiomelanocortin (POMC) gene regulation in stably transfected AtT-20 cells overexpressing LIF.

Leukemia inhibitory factor (LIF) levels are elevated in sepsis and correlate with shock and poor prognosis. We have previously shown that lipopolysaccharide (LPS) administration induces hypothalamic and pituitary LIF expression in vivo, which is associated with the acute rise in circulating adrenocorticotrophic hormone (ACTH) levels. As AtT-20 cells respond to LIF, we established murine LIF (mLIF) stably transfected AtT-20 cell lines to study LIF regulation of pro-opiomelanocortin (POMC) expression and ACTH secretion. Our results show that mLIF transfectants accumulated mLIF (up to 15.6 +/- 3.2 ng/mL after 24 h) as well as increased ACTH secretion (up to 2.4-fold above control cells) in conditioned medium. The magnitude of ACTH induction correlated with mLIF concentrations in different transfectants (r = 0.75-0.88, p < 0.05). Moreover, mLIF transfectants showed a higher sensitivity to CRH stimulation with an increased ACTH production within 8 h (p < 0.05), whereas control cells were responsive to CRH at 24 h. Additionally, mLIF transfectants exhibited a maximum threefold ACTH induction, compared to 1.7-fold in control cells. Furthermore, mLIF transfectants have a blunted dexamethasone-mediated inhibition of ACTH (35% inhibition in control cells vs no inhibition in mLIF-transfected cells at 24 h). These findings support and extend the previous observations of LIF acting at the pituitary level, and indicate that mLIF stably-transfected AtT-20 cells are a useful model for studying mLIF-mediated gene regulation in pituicytes.

Adrenocorticotropic Hormone↗
PubMed2003

Impaired thyroglobulin (Tg) secretion by FRTL-5 cells transfected with soluble receptor associated protein (RAP): evidence for a role of RAP in the Tg biosynthetic pathway.

Secretion of thyroglobulin (Tg) by thyrocytes requires several endoplasmic reticulum (ER)-resident molecular chaperones. The receptor-associated protein (RAP), a known molecular chaperone, binds to Tg in thyroid cells shortly after biosynthesis. Here we investigated whether RAP is involved in Tg secretion by FRTL-5 cells. For this purpose, we studied Tg secretion by FRTL-5 cells transfected with a soluble RAP chimera, as a mean for interfering with endogenous RAP. We used a RAP-human IgG Fc (RAP-Ig) chimeric cDNA, which was designed in order to exclude the ER retention sequence of RAP and to allow generation of a secreted form of RAP. FRTL-5 cells were transiently transfected with the RAP-Ig cDNA or, as control, with a CD8-Ig cDNA. Media were collected at 24, 48 and 72 h after transfection. Secretion of fusion proteins and of Tg in the media was measured by ELISA. As expected, under standard culture conditions, RAP was not secreted into the media by FRTL-5 cells, even though it could be detected by Western blotting in cell extracts. In transfection experiments, fusion proteins were present in the media of FRTL-5 cells transfected with either RAP-Ig or CD8-Ig, indicating that transfection was successful. Although Tg was found in the media of FRTL-5 cells transfected with either CD8-Ig or RAP-Ig, a lower amount was found in cells transfected with RAP-Ig. Therefore, we concluded that RAP is involved in Tg secretion by FRTL-5 cells suggesting that RAP may function as a Tg molecular chaperone.

Blotting, Western↗
PubMed2004

Improvement of transfection efficiency of epithelioma papulosum cyprini carp cells by modification of cell cycle and use of an optimal promoter.

Several methods to improve transfection of epithelioma papulosum cyprini (EPC) carp cells have been tested and are reported here. By modifying the cell cycle state of EPC cell monolayers and selecting the best promoter for the plasmid to be transfected, we increased transfection efficiency from 12.8% to 55.1% and decreased the coefficient of variation among different experiments from 54.1% to 11.8%. Thus 2- to 3-fold higher transfection efficiencies were obtained when the EPC monolayers were treated with colchicine or thymidine before transfection. In addition, the plasmids pMOKbetagal and its shorter derivative pMVC1.4betagal, both containing 218 bp of additional sequences upstream of the cytomegalovirus promoter contained in plasmid pCMVbeta, consistently produced higher transfection efficiencies than pCMVbeta. Combination of the two methods resulted in an improvement of both efficiency and reproducibility. These results should facilitate transfection of EPC cells to use as a model to obtain transgenics, to conduct quantitative transfected-cell fusion assays, to improve DNA-immersion-vaccination methods, or to obtain infectious cDNA from fish RNA viruses.

Animals↗
PubMed1991

Lipophilic polylysines mediate efficient DNA transfection in mammalian cells.

Low molecular weight (Mr approximately 3000) poly(L-lysine) (PLL) conjugated to N-glutarylphosphatidylethanolamine is an effective carrier to promote DNA-mediated transfection in cultured mammalian cells. The conjugates, named 'lipopolylysines', contained an average of two phospholipid groups per molecule of PLL. Similar conjugates of the non-degradable poly(D-lysine) also had a similar transfection activity, indicating that the degradation of the carrier is not required for the activity. Unconjugated polylysines had little activity. The transfection activity of the lipopolylysine has been optimized with respect to the DNA concentration, DNA/carrier ratio, incubation time and the presence of serum in the incubation medium. The binding of lipopolylysine with DNA was measured by the degree of retardation of DNA in agarose gel electrophoresis. It was found that at the optimal DNA/lipopolylysine ratio for transfection, all DNA were found in large complexes which did not enter the gel. The transfection activity of the lipopolylysine, under optimal conditions, was approximately 3-fold higher than that of lipofectin, a widely used commercial reagent. Moreover, lipopolylysine mediated transfection even in the presence of 10% calf serum; whereas the lipofectin lost about 70% of its activity under the same condition. However, unlike lipofectin the transfection activity of the lipopolylysine depended on scraping the treated cells. Furthermore, lipopolylysine only transfected attached monolayer cells, and not suspension cells.

Animals↗
PubMed1994

A2a/D2 receptor interactions are not observed in COS-7 cells transiently transfected with dopamine D2 and adenosine A2a receptor cDNA.

The rat D2 receptor and the dog A2a receptor subcloned into the pXM vector were transiently transfected into COS-7 cells using the DEAE-dextran method. The transfected cells expressed approx. 200 fmol D2 receptors/mg protein and approx. 5 pmol/mg protein of the A2a receptor as judged by binding experiments with [3H]raclopride [or[3H]-N-propyl-apomorphine (NPA)] and [3H]-CGS 21680, respectively. The high affinity KD values were 0.43 and 19 nM for D2 and A2a receptors, respectively, in agreement with results obtained from other cells and tissues. The non-selective adenosine receptor agonist NECA stimulated cAMP accumulation both in non-transfected and transfected COS-7 cells with only a slight difference in potency, suggesting that most of the stimulation is due to activation of A2b receptors known to be present on virtually every cell. The two A2a selective agonists CGS 21680 and CV-1808 were essentially inactive in transfected COS-7 cells, but were very active in PC-12 cells known to possess functional A2a receptors. Dopamine did not decrease cAMP accumulation in the transfected COS-7 cells. CGS 21680 (30 nM) did not affect the binding characteristics of D2 receptors in the co-transfected COS-7 cells in contrast to the increased KH, KL and RH values found previously in rat striatal membranes after CGS 21680 treatment. The present findings indicate that transiently transfected A2a and D2 receptors in COS-7 cells have normal binding properties, but couple poorly to adenylyl cyclase, despite the presence of Gs protein and adenylyl cyclase in these cells. Our results also demonstrate that the previously reported interactions between A2a receptors and D2 receptors do not occur when only the receptor proteins are expressed in COS-7 cells, suggesting that the two receptor molecules do not interact directly to influence binding characteristics.

Adenosine↗
PubMed1996

Selective isolation of transiently transfected cells from a mammalian cell population with vectors expressing a membrane anchored single-chain antibody.

We present here a novel technology for the rapid selection of transiently transfected cells from total populations in culture. This system utilizes recombinant antibody technology to produce a 'molecular hook' by displaying a hapten-binding single-chain antibody (sFv) on the surface of transfected cells. Mammalian cell lines from several origins were transiently transfected with a plasmid (pHook-1) that encodes an sFv fused with a transmembrane anchor and found to express and display the functional hapten-binding sFv on their membranes. Transfected cells were selected from total populations in culture by virtue of their ability to bind to hapten-coated magnetic beads. Some cell lines were able to display sFv sufficient for selection as early as 2 h post-transfection. SK-BR-3 human breast carcinoma cells were co-transfected with pHook-1 and pCR31acZ (expresses beta-galactosidase), selected, and assayed for beta-galactosidase activity. The positive correlation between sFv and beta-galactosidase expression in these cells (95% of selected cells also expressed beta-galactosidase activity) suggests that pHook-1 will be useful in isolating cells co-expressing an exogenous gene of interest. Another vector was constructed in which a gene of interest may be expressed from the same plasmid as the sFv 'hook'. This construct (pHook-2) allows the selection of a homogenous population of cells expressing exogenous genes without co-transfection or the generation of stable transfectants. In experiments where the lacZ gene was co-expressed with the sFv 'hook' from this single plasmid, 100% of 293 human kidney cells and 100% of SK-BR-3 cells selected with antigen-coated magnetic beads stained positively for beta-galactosidase activity. We propose that this system will be a valuable tool for studying the acute and chronic effects of the expression of a variety of wild type and mutant proteins.

Amino Acid Sequence↗
PubMed1987

Unusual expression of HLA molecules at the surface of murine cells transfected with HLA-B7 or HLA-A11 genes.

The HLA-B7 and HLA-A11 molecules expressed on murine transfectants have been analysed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). Two different murine cells, L and P815-HTR have been compared, because it has been previously established that P815 transfectants were much more sensitive to human cytolytic cells than L transfectants. Three kinds of HLA molecules were present on these cells: (1) normal HLA molecules with 2D-PAGE profiles identical to those of the molecules isolated from human cells; (2) HLA molecules of usual size but with more various charges than HLA molecules detected on human cells. This heterogeneity was constantly found with cells expressing HLA-B7 or -A11 antigens, both in L and in P815 transfectants, including several clones. These forms were detected by anti-HLA monoclonal antibodies and by antipeptide (from HLA-B7) antibodies; (3) other unusual products corresponding to shorter heavy chains: molecules of various mol. wts and charges were detected in HLA-B7 but not in HLA-A11 transfectants. They were observed using antipeptide sera but were not seen with anti-HLA monoclonal antibodies. These products were possibly related to the DNA used for transfection and it cannot be excluded that such abnormalities only detectable by antipeptide sera would exist in other transfectants. The functional discrepancies between P815 and L transfectants cannot be clearly explained by these biochemical results.

Animals↗