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Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line.

Abbasi-Kenarsari H, Shafaghat F, Baradaran B, Movassaghpour AA, Shanehbandi D, Kazemi T - Avicenna J Med Biotechnol (2015 Jan-Mar)

Bottom Line: PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria.After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing.NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent.

View Article: PubMed Central - PubMed

Affiliation: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran ; Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran ; Students Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.

ABSTRACT

Background: CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study.

Methods: Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic.

Results: Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively).

Conclusion: Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera.

No MeSH data available.


Related in: MedlinePlus

Flow cytometric analysis of expression of human CD19. Using specific monoclonal antibody, expression of human CD19 for transient (A) and stable expression (B); untransfected NIH-3T3 (C); mock-transfected NIH-3T3 (D); and Raji cell lines (E, as positive control) were analyzed.
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Figure 0003: Flow cytometric analysis of expression of human CD19. Using specific monoclonal antibody, expression of human CD19 for transient (A) and stable expression (B); untransfected NIH-3T3 (C); mock-transfected NIH-3T3 (D); and Raji cell lines (E, as positive control) were analyzed.

Mentions: NIH-3T3 mouse fibroblastic cell line was transfected by pCMV6-Neo/CD19 recombinant expression vector using JetPEI transfection reagent and transient expression of CD19 was confirmed by surface flow cytometry 48 hr after transfection (Figure 3A). After that, pCMV6-Neo/CD19- and mock-transfected and also untransfected NIH-3T3 cells were subjected to growth in complete cell culture medium containing 400 µg/ml G418 which was the minimal dose for elimination of untransfected NIH-3T3 cells. After 3 days, untransfected cells died and transfected cells continued to grow in G418 up to 1500 µg/ml during two months. Cells were finally analyzed by surface flow cytometry and significant positive reactivity of pCMV6-Neo/ CD19- transfected cells was observed (93%) (Figure 3B). On the other hand, untransfected and mock-transfected NIH-3T3 cells showed no positive signal in flow cytometry (4 and 6%, respectively) (Figures 3C and D).


Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line.

Abbasi-Kenarsari H, Shafaghat F, Baradaran B, Movassaghpour AA, Shanehbandi D, Kazemi T - Avicenna J Med Biotechnol (2015 Jan-Mar)

Flow cytometric analysis of expression of human CD19. Using specific monoclonal antibody, expression of human CD19 for transient (A) and stable expression (B); untransfected NIH-3T3 (C); mock-transfected NIH-3T3 (D); and Raji cell lines (E, as positive control) were analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388889&req=5

Figure 0003: Flow cytometric analysis of expression of human CD19. Using specific monoclonal antibody, expression of human CD19 for transient (A) and stable expression (B); untransfected NIH-3T3 (C); mock-transfected NIH-3T3 (D); and Raji cell lines (E, as positive control) were analyzed.
Mentions: NIH-3T3 mouse fibroblastic cell line was transfected by pCMV6-Neo/CD19 recombinant expression vector using JetPEI transfection reagent and transient expression of CD19 was confirmed by surface flow cytometry 48 hr after transfection (Figure 3A). After that, pCMV6-Neo/CD19- and mock-transfected and also untransfected NIH-3T3 cells were subjected to growth in complete cell culture medium containing 400 µg/ml G418 which was the minimal dose for elimination of untransfected NIH-3T3 cells. After 3 days, untransfected cells died and transfected cells continued to grow in G418 up to 1500 µg/ml during two months. Cells were finally analyzed by surface flow cytometry and significant positive reactivity of pCMV6-Neo/ CD19- transfected cells was observed (93%) (Figure 3B). On the other hand, untransfected and mock-transfected NIH-3T3 cells showed no positive signal in flow cytometry (4 and 6%, respectively) (Figures 3C and D).

Bottom Line: PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria.After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing.NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent.

View Article: PubMed Central - PubMed

Affiliation: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran ; Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran ; Students Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.

ABSTRACT

Background: CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study.

Methods: Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic.

Results: Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively).

Conclusion: Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera.

No MeSH data available.


Related in: MedlinePlus