Antibiotic G418 Research Articles

Establishment of an efficient genetic transformation system for Tanacetum cinerariifolium

The Dalmatian Daisy Tanacetum cinerariifolium is an Asteraceae plant species that produces the natural insecticide “pyrethrum”, which is effective against mosquito disease vectors and household pests. To enhance the content of pyrethrum in flowers, a more detailed understanding of the mechanisms underlying pyrethrum biosynthesis is needed. Even though gene transformation and genome editing techniques are vital for investigating pyrethrin biosynthesis, limited information is available on the transformation of T. cinerariifolium. Furthermore, each seedling possesses a distinct genotype with large variations by self-incompatibility. We herein employed T. cinerariifolium line #14 with weak self-incompatibility to establish a protocol of efficient regeneration from leaf segments and transformation. Leaf segments formed calli on 1/2 Murashige and Skoog’s basal medium (MS) with naphthalene acetic acid 1 mg L−1 and 6-benzylaminopurine (BAP) 2 mg L−1, regenerated shoots from calli on 1/2 MS with BAP 0.5 mg L−1 and GA3 0.2 mg L−1, and elongated shoot stems on 1/2 MS with indole-3-butyric acid 0.5 mg L−1 and BAP 0.5 mg L−1. To establish genetic transformation, Rhizobium radiobacter strain EHA105 with the highest infectivity and the mas1'-2' bidirectional promoter with the highest expression of the nptII resistance gene were used, and the antibiotic G418 was added to medium at a concentration of 10 to 20 mg L−1 to select transformed cells. Using established regeneration techniques, we successfully obtained transformants that highly expressed the transgene gusA. This technique will be useful for creating genetically modified T. cinerariifolium, particularly for elucidating the mechanism of pyrethrin biosynthesis toward the creation of pyrethrin-rich traits.

A new miniMOS tool kit capable of visualizing single copy insertion in C. elegans.

The miniMOS technique has been widely used in the C. elegans community to generate single copy insertions. A worm is considered as a potential insertion candidate if it is resistant to G418 antibiotics and does not express a co-injected fluorescence marker. If the expression of the extrachromosomal array is very low, it is possible for a worm to be mistakenly identified as a miniMOS candidate, as this low expression level can still confer resistance to G418 without producing a detectable fluorescence signal from the co-injection marker. This may increase the workload for identifying the insertion locus in the subsequent steps. In the present study, we modified the plasmid platform for miniMOS insertion by incorporating a myo-2 promoter-driven TagRFP or a ubiquitous H2B::GFP expression cassette into the targeting vector and introducing two loxP sites flanking the selection cassettes. Based on this new miniMOS tool kit, the removable fluorescence reporters can be used to visualize the single copy insertions, greatly reducing insertion locus identification efforts. In our experience, this new platform greatly facilitates the isolation of the miniMOS mutants.

The Hydrophobicity and Antifungal Potentiation of Burkholdine Analogues.

The burkholdines are a family of cyclic lipopeptides reported to exhibit antifungal activity. We synthesized a series of 18 burkholdine analogues in good yield by conventional Fmoc-SPPS followed by cyclization with DIPCI/HOBt in the solution phase. Although none of the synthesized peptides exhibited antifungal activity, several did potentiate the antibiotic effect of the antibiotic G418, including the Thr-bearing Bk analogue (4b) and the tartaramide-bearing Bk analogue (5b). This work exemplifies the potential of burkholdine analogues as potentiating agents.

Choice of selectable marker affects recombinant protein expression in cells and exosomes

Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.

Sialic acid sulfation is induced by the antibiotic treatment in mammalian cells.

Sialic acids (Sias) are an outermost-situated sugar of glycoproteins and glycolipids to play important roles in various biological phenomena. They are often modified by additional substituents, such as O-acetyl group, to display more than 50 different structures in nature. Of those modified Sia, nothing is known about the occurrence and biological functions of sulfated Sias (SiaSs) in mammals. To elucidate the significance of sialic acid sulfation, we investigated various mammalian-cultured cell lines for the expression of SiaS using the specific antibody 3G9. First, SiaS is expressed in a cell line-dependent and a cell density-dependent manner. Second, in CHO cells, the expression of SiaS is reversibly induced by treatment with the antibiotic G418. Taken together, the expression of SiaS is changed by intrinsic and extrinsic factors in mammalian cells. This is the first demonstration of regulated expression of SiaS.

Suppressing 'nonsense' in cystic fibrosis.

Suppressing 'nonsense' in cystic fibrosis.

Change in the Expression of BAK، FAS، BAX، TNF-A، BCL-2 and Survivin Apoptosis Genes of the Human Breast Adenocarcinoma Cells (MCF-7) by Staphylococcal Enterotoxin B

Introduction: Breast cancer is one of the most prevalent malignancies among women. Patients whose suffering from this condition, as a result of the use of conventional therapies often have a poor response to treatment and the relapse among them is frequent. In this study, the effects of staphylococcal enterotoxin type B on BAK، FAS، BAX، TNF-a، BCL-2 و Survivin genes expression in human breast adenocarcinoma cells (MCF-7) was examined. Staphylococcal enterotoxin B is a powerful member of the Staphylococcus aureus toxins family, which is known as an anticancer agent with potential for killing cancer cells. Methods: The experimental study was carried out at the Biotechnology Research Center of Islamic Azad University, Shahrekord Branch. By using Lipofectamine 2000 reagent, MCF-7 cells transfected with the pcDNA3.1(+)-seb (recombinant) and pcDNA3.1(+) (non-recombinant) plasmids and were selected by culturing in a selective medium of RPMI- 1640 containing 600 μg / mL antibiotic G418. Then, the expression of BAK, FAS, BAX, TNF-a, BCL-2, and Survivin genes in transfected cells were analyzed by real time PCR. Student's t-test, SPSS Inc., Chicago, IL; Version 16 and also Excel program for statistical analysis were used. Results: The results of this study indicated that staphylococcal enterotoxin type B (SEB) remarkably changes the expression of apoptotic related genes in MCF-7 cell line. It was observed a significant increase in the expression of BAK, FAS, BAX, and TNF-a genes, the expression of BCL-2 and Survivin genes significantly decreased compared to the control group (P=0/032). Conclusion: Staphylococcal enterotoxin type B has an inhibitory effect on the growth, proliferation and invasion of breast adenocarcinoma cells through altering the expression of the genes involved in the apoptosis process. Therefore, it seems that there is a good research field for the use of this toxin in the control and treatment of human breast adenocarcinoma.

Expression of Human Erythropoietin Containing 2 Additional N-Link in CHO-K1 Cells under Different Culture Conditions

Human erythropoietin (hEPO) is a glycoprotein that regulates the formation of erythrocytes and mainly used in anemia patients. Previously, we have reported the expression of modified human EPO with 2 additional N-linked in mammalian cell CHO-K1. The aim of this current research was to study the optimum condition for modified recombinant hEPO (rhEPO) production in CHO-K1. To do this, several parameters of culture conditions were applied including antibiotic concentrations, seeding densities, time of incubations, fetal bovine serum (FBS) concentrations and cell culture media. The result showed that the presence of antibiotic G418 improved the expression level with the highest was at 1% of concentration. Meanwhile, seeding density of 2–3x105 cells/6 cm dish and seven day of incubation time were the best condition for rhEPO protein expression. From five different combination media used, F12 medium with 10% FBS gave the highest expression of rhEPO protein. From this study was also found that at passage 16 the expression level was still increasing proving that the clone expressing the protein of our interest is promisingly stable.Keywords : EPO, erythropoietin, protein expression, CHO-K1, optimation

High level expression and purification of recombinant human serum albumin in Pichia pastoris

Human serum albumin (HSA) has been extensively used in a series of clinical care settings for nearly seven decades. However, the broad application of this protein is seriously limited by its short supply. In this work, the codon sequence of HSA was cloned under the control of the alcohol oxidase 1 promoter (AOX1) and expressed as a secretory protein in Pichia pastoris. A recombinant strain displaying the highest HSA yield was selected by screening for resistance to the highest concentration of antibiotic G418. After optimizing the induction conditions and additional supplements, the highest yield of HSA reached 1.6 g/L in a shake flask. Performing high density fermentation further improved the highest yield to 8.86 g/L in a fermenter after 96 h of methanol induction. This result is more promising than the previous reports of industrial applications, which reported the highest yield as 92.29 mg/L/h, considering that the space-time yield of rHSA was doubled. In addition, the desired protein was purified by filtration and Cibacron Blue affinity chromatography, which yielded a 58% recovery of a product that had over a 96% purity. This study reveals that Pichia pastoris is an excellent system for recombinant human serum albumin expression due to its outstanding expression capacity. In addition, the high efficiency level of rHSA production lays a solid foundation for its use in industrial production.

Lipocalin2 Protects Human Embryonic Kidney Cells against Cisplatin-Induced Genotoxicity.

Cisplatin is one of the most useful chemotherapeutics which performs its cytotoxic effect via accumulation of platinum resulting in oxidative stress, and destruction of cell DNA. This could probably cause secondary cancers in healthy tissues. Lipocalin2 (Lcn2) is a protein which its expression is increased in oxidative stresses. Therefore, the present study was performed to evaluate the protective effects of Lcn2 up-regulation on cisplatin genotoxicity. In order to up-regulate Lcn2 expression, HEK293 cells were transfected with pcDNA3.1-Lcn2 vector. Afterwards, stable cells consistently expressing Lcn2 were selected via screening with G418 antibiotic. Next, overexpression of Lcn2 was evaluated by RT-PCR and ELISA, comparing to the control non-transfected cells. Then, in order to evaluate the cytoprotective effects of Lcn2 overexpression, transfected and non-transfected cells were subjected to cisplatin treatment followed by MTT and alkaline Comet assays. RT-PCR and ELISA assays confirmed up-regulation of Lcn2 by the stable cells. MTT assay of the Lcn2 over-expressing cells showed higher IC50 values comparing to the non-transfected cells. Furthermore, the Comet assay confirmed Lcn2 protective effects on the cisplatin (1 µg/mL) induced genotoxicity. In the present study, for the first time, we showed the protective effect of Lcn2 on cisplatin induced genotoxicity. Therefore, one of the probable mechanisms of Lcn2 cytoprotctive effects under oxidative stress conditions could be due to the prevention of genotoxicity. However, further evaluations in this regard must be considered.

Construction and high-density fermentation of alkaline pectate lyase high-yield yeast

Pectate lyase is widely applied in ramie degumming and fabric bioscouring in the textile industry. Compared to conventional processes that involve high alkaline and high temperature treatment, enzyme based treatments have significant advantages in fibers protectiveness, improved efficiency of refining, reduced energy consumption and pollution. Hence, it would be highly desirable to construct high-yield alkaline pectate lyase engineered strains and reduce the pectate lyase production cost. In the previous study, pectate lyase gene pel from Bacillus subtilis168 was expressed in Pichia pastoris GS115 after codon usage optimization based on the vector pHBM905A. To improve the expression level, the vector pHBM905BDM with optimized promoter and signal peptide was used to express the optimized gene pels in GS115. The transformant had increased activity from 68 U/mL to 100 U/mL with the improvement in the transcription level by 27% measured by qPCR. The transformants were further screened on pectin plates, where higher halo forming strains were picked for shake-flask fermentation and strain GS115-pHBM905BDM-pels4 showed the highest activity of 536 U/mL. Then plasmid pPIC9K-pels was constructed and electroporated into the GS115-pHBM905BDM-pels4 cells. Subsequently, high-copy transformant was screened by using the medium containing antibiotics G418, strain GS115-pHBM905BDMpPIC9K- pels1 was identified with increased activity of 770 U/mL and the copy number of pels was 7 confirmed by qPCR. Finally, the activity of pectate lyase produced by GS115-pHBM905BDM-pPIC9K-pels1reached to 2 271 U/mL in a 5-L fermentor. The activity of pectate lyase in our study reached the highest level of expression in P. pastoris, showing good application potential in the textile industry.

Genetic transformation of the white-rot fungus Dichomitus squalens using a new commercial protoplasting cocktail

D. squalens, a white-rot fungus that efficiently degrades lignocellulose in nature, can be used in various biotechnological applications and has several strains with sequenced and annotated genomes. Here we present a method for the transformation of this basidiomycete fungus, using a recently introduced commercial ascomycete protoplasting enzyme cocktail, Protoplast F. In protoplasting of D. squalens mycelia, Protoplast F outperformed two other cocktails while releasing similar amounts of protoplasts to a third cocktail. The protoplasts released using Protoplast F had a regeneration rate of 12.5% (±6 SE). Using Protoplast F, the D. squalens monokaryon CBS464.89 was conferred with resistance to the antibiotics hygromycin and G418 via polyethylene glycol mediated protoplast transformation with resistance cassettes expressing the hygromycin phosphotransferase (hph) and neomycin phosphotransferase (nptII) genes, respectively. The hph gene was expressed in D. squalens using heterologous promoters from genes encoding β-tubulin or glyceraldehyde 3-phosphate dehydrogenase. A Southern blot confirmed integration of a resistance cassette into the D. squalens genome. An average of six transformants (±2 SE) were obtained when at least several million protoplasts were used (a transformation efficiency of 0.8 (±0.3 SE) transformants per μg DNA). Transformation of D. squalens demonstrates the suitability of the Protoplast F cocktail for basidiomycete transformation and furthermore can facilitate understanding of basidiomycete gene function and development of improved strains for biotechnological applications.

English

Gua1 gene in Schizosaccharomyces pombe encodes inosine 5′-monophosphate dehydrogenase (IMPDH), which catalyzes the first step in de novo biosynthesis of guanosine monophosphate (GMP). Knock-out cassette was constructed with polymerase chain reaction (PCR)-based gene targeting technique for deletion of gua1 gene in S. pombe and this knock-out cassette was transformed to S.s pombe wild type (972h-). Knock-out cassette contains 653 and 285 bp sequences which are upstream and downstream of gua1 gene, respectively, in S. pombe genome and kanamycin resistance gene obtained from pFA6 plasmid. After transformation using lithium acetate method, knock-out cassette is aimed at replacing with the sequences of gua1 gene via homologous recombination. The transformant 972h- colonies which integrated knock-out cassette to the genome via homologous recombination are selected in YEA medium with antibiotic G418 after transformation and in this step, possible mutant colonies in gua1 gene were determined. Finally, colony PCR techniques were performed to control whether the deletion is made in the right place or not. The results show that the gua1 gene deleted strain was obtained.   Key words: Fission yeast, deletion, gua1 gene, knock-out cassette, inosine 5′-monophosphate dehydrogenase (IMPDH).

High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

The acetylcholinesterase 2 (AChE2) cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115. One transformant with high-level expression of the recombinant AChE (rAChE, 23.2 U mL−1 in supernatant) was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate. The maximum production of rAChE reached about 11.8 mg of the enzyme protein per liter of culture. The rAChE was first precipitated with ammonium sulfate (50% saturation) and then purified with procainamide affinity column chromatography. The enzyme was purified 12.1-fold with a yield of 22.8% and a high specific activity of 448.3 U mg−1. It was sensitive to inhibition by methamidophos and pirimicarb, the calculated 50% inhibitory concentration (IC50) values of the two pesticides were 0.357 and 0.888 mg L−1, respectively, and the calculated 70% inhibitory concentration (IC70) values were 0.521 and 0.839 mg L−1, respectively. The results suggested that it has a potential application in the detection of pesticide residues.

Cav‐1 Upregulation Slows Migration Velocity in BLM Melanoma

Caveolin‐1 (Cav‐1) is a membrane‐associated and cytosolic protein that influences a number of cellular signaling cascades. Changes in Cav‐1 expression levels are thought to be responsible for changes in metastatic behaviour of a number of cancers, including melanoma. However, the effect of changes in Cav‐1 expression level seem dependent on cell‐type and direction of expression change. In highly migratory, metastatic melanoma, Cav‐1 expression levels tend to be low. The purpose of this study was to determine whether Cav‐1 upregulation alters EMT markers, cell division or basal and CXCL‐8‐stimulated migration velocity in human BLM melanoma. Wild‐type BLM cells were stably transfected (Lipofectamine 2000, Invitrogen) with a Myc‐DDK‐tagged human Cav‐1 gene cloned into a pCMV6‐Entry vector (Origene, TrueOrf). Permanent selection was achieved with G418 (1 mg/ml) antibiotic. Cell extracts from wild‐type and transfected BLM cells were run on 10%‐PAGE gels and transferred to PVDF membranes. Appropriate 1° antibodies were used to confirm Cav‐1 upregulation and to test for the presence of the EMT marker N‐cadherin using infrared 2° antibody detection (LiCor). Proliferation of wild‐type and Cav‐1 transfected cells were measured using BrdU assay. Cells were plated in 96‐well plates and incubated for 24 hours in RPMI 1640, 10% FBS. BrdU was added to the cells and allowed to incubate for 12 hours. Cells were fixed, the DNA denatured, and mouse anti‐BrdU antibody added to detect the incorporated BrdU. BrdU uptake was measured using HRP‐linked 2° antibody and [TMB] detected at A450 nm using a spectrophotometer (Epoch Microplate Spectrophotometer). Migration velocity was measured in wild‐type and transfected cells using a wound‐healing assay. Cells were seeded at 250,000 cells per well in 24‐well plates and incubated overnight in serum free RPMI 1640 media. Wells were scratched with pipette tip and plated in RPMI 1640, 10% FBS. Wells received a range of unbound CXCL8 doses (0–100 ng/ml). Wound images were collected at 15 min intervals and quantified at 4 hour intervals using ImageJ. Immunoblot analysis confirmed increased expression of Myc‐DDK‐tagged Cav‐1 in the transfected BLM cells. Immunohistochemical staining also confirmed increased expression of Cav‐1 in the transfected cells and showed that subcellular distribution of Cav‐ 1 is similar in the wild‐type and transfected cells. The EMT marker N‐cadherin, which was largely absent in the wild‐type cells, showed increased expression in the Cav‐1 transfected BLMs. BrdU assay shows that there is no significant difference in cell division over 12 hours between wild‐type and Cav‐1 transfected cells by Student's t‐test (p = 0.54). Migration velocity by wound‐closure assay decreased with Cav‐1 overexpression. Wild‐type cells migrated at 14.0 ± 4.9 μm / h (mean ± sd). Transfected cells migrated at 7.0 ± 2.2 μm / h. This was statistically significant by Student's t‐test (p < 0.001). In both wildtype and Cav‐1 transfected BLM melanoma, we were unable to demonstrate altered cell migration rates when unbound CXCL8 was administered over a 24 hour period in RPMI media containing 10% FBS. In summary, Cav‐1 expression in BLM melanoma is associated with an increased N‐cadherin expression, no change in cell division by BrdU assay, and slowed migration by wound‐healing assay. More work will be necessary to elucidate the underlying mechanisms.Support or Funding InformationSupported by the Graduate Program Committee, KCOM

Evidence of phenotypic stability after transduction of fluorescent proteins in two human tongue cancer cell lines

ObjectivesThis study investigated the phenotypic stability and biological properties of two human tongue cancer cell lines after transduction of fluorescent proteins. DesignThe human tongue cancer cell lines UM1 and UM2 were cultured with GFP and RFP lentiviral particles stock for 72h. Cells with successful transduction of fluorescent proteins were selected in a medium containing G418 antibiotics for two weeks. The proliferation rates of parental and transduced cell lines were evaluated by their population doubling time (PDT). Transduction efficiency was assessed by fluorescence microscope and flow cytometry. The transduced cells in passage 1, 2, 10, 20 and 30 were collected to check the stability of fluorescent protein expression. Phenotypic stability of the transduced cells was detected by means of cell morphology, cell surface markers and cell function evaluating essay. ResultsThe proliferation rates of the transduced cell lines showed no significant difference compared to their parental cells. Successful transduction with high efficiency (99% up) was demonstrated. High fluorescence expression on both transduced cells was detected until the thirtieth generation. UM1 and UM1-GFP displayed mesenchymal cell characteristics, while UM2 and UM2-RFP cell lines showed properties characteristic of epithelial. ConclusionsTwo human tongue cancer cell lines of epithelial and mesenchymal phenotype respectively, have been successfully labelled with green and red fluorescent proteins. The fluorescence maintained a high expression rate over thirty generations without influencing the original morphological phenotype and cadherin expression.

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol.

The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains. Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, Schizosaccharomyces pombe. In the following we describe how to swap the ura4+ marker to a kanMX6, natMX4, or hphMX4 marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the MX markers for nutritional markers, such as arg3+, his3+, leu1+ and ura4+ .

Sensitivity of McCoy-Plovdiv cells to G-418 antibiotic

Sensitivity of McCoy-Plovdiv cells to G-418 antibiotic

Generation of CHO Stable Cell Line Overexpressing HER2: an In Vitro Model for Breast Cancer

DOI : 10.7508/rmm.2015.03.002 Abstract Background: Breast cancer is the most common female malignancy and the leading cause of cancer mortality in women worldwide. The human epidermal growth factor receptor2 (HER2) is a transmembrane tyrosine kinase receptor that is usually overexpressed in human breast cancers. Stable cell lines heterogeneously overexpressing HER2 are highly required as in vitro models for breast cancer research. The aim of this study was to establish a stable cell line overexpressing HER2. Materials and Methods: CHO Cells were transfected with linearized pCVN/HER2 plasmid and selected for the recombinant cells with G418 antibiotic. Expression of HER2 in the transfected cells was analyzed using western blotting and immunofluorescence. Results: We found that the recombinant cells stably expressed high levels of HER2 proteins that were mostly concentrated on the cell membrane. Conclusions: The cell line established here provides a useful in vitro model for breast cancer research and any HER-related studies.

Cloning and Stable Expression of cDNA Coding For Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1, CD31) in NIH-3T3 Cell Line.

PECAM-1 (CD31) is a glycoprotein expressed on endothelial and bone marrow precursor cells. It plays important roles in angiogenesis, maintenance and integration of the cytoskeleton and direction of leukocytes to the site of inflammation. We aimed to clone the cDNA coding for human CD31 from KG1a for further subcloning and expression in NIH-3T3 mouse cell line. CD31 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. 2235 bp specific band was aligned completely to human CD31 reference sequence in NCBI database. Transient and stable expression of human CD31 on transfected NIH-3T3 mouse fibroblast cells was achieved (23% and 96%, respectively) as shown by flow cytometry. Due to murine origin of NIH-3T3 cell line, CD31-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD31, with no need for purification of recombinant proteins.