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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

Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker (bp). Asterisks (*) point the desired band.
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Figure 0001: Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker (bp). Asterisks (*) point the desired band.

Mentions: Flow cytometric analysis of Raji cells, as the source for amplification of CD19 cDNA and positive control for surface expression of CD19 in further experiments, showed positive reactivity with anti-CD19 monoclonal antibody (M2: 98%) in comparison with cells stained by isotype control (M1: 2%). On the other hand, expression of CD19 mRNA was successfully showed by amplification of CD19 cDNA in PCR method (Figure 1A), as described later.


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)

Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker (bp). Asterisks (*) point the desired band.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0001: Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker (bp). Asterisks (*) point the desired band.
Mentions: Flow cytometric analysis of Raji cells, as the source for amplification of CD19 cDNA and positive control for surface expression of CD19 in further experiments, showed positive reactivity with anti-CD19 monoclonal antibody (M2: 98%) in comparison with cells stained by isotype control (M1: 2%). On the other hand, expression of CD19 mRNA was successfully showed by amplification of CD19 cDNA in PCR method (Figure 1A), as described later.

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