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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

Alignment of amplified cDNA for canonical isoform of human CD19 reference sequence in NCBI database. Comparing the 1701 bp amplified sequence with reference sequence for short isoform (variant 2) of human CD19 showed complete alignment. Only 5‘ and 3‘ of alignment has been briefly showed.
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Figure 0002: Alignment of amplified cDNA for canonical isoform of human CD19 reference sequence in NCBI database. Comparing the 1701 bp amplified sequence with reference sequence for short isoform (variant 2) of human CD19 showed complete alignment. Only 5‘ and 3‘ of alignment has been briefly showed.

Mentions: Initial optimization using Taq DNA polymerase showed specific band in annealing temperature of 64°C and 1 mM concentration of MgCl2. Subsequent PCR reaction using Pfu DNA polymerase led to a single band with expected size (1701 bp) (Figure 1A). PCR product was ligated into pGEM-T Easy vector by TA-cloning method and then transformed to competent DH5α bacteria using heat shock method. Blue-white selection was used for screening white colonies containing recombinant plasmid. Among them, eight white colonies were subjected to colony-PCR with gene-specific primers and two of them showed the expected band (Figure 1B). Then, miniprep was prepared from single positive colony and double digested with KpnI and HindIII restriction enzymes. Because of existence of one restriction site for KpnI in CD19 gene at nucleotide position 603, pGEM-T Easy-CD19 construct was subjected to partial digestion by KpnI and complete digestion by HindIII and led to excision of insert with length of 1701 bp (Figure 1C). Alignment of sequencing result with reference sequence in NCBI database showed complete matching with CD19 mRNA isoform 2 except at nucleotide position 4 where C was replaced with G in order to improve the expression of gene (Figure 2).


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)

Alignment of amplified cDNA for canonical isoform of human CD19 reference sequence in NCBI database. Comparing the 1701 bp amplified sequence with reference sequence for short isoform (variant 2) of human CD19 showed complete alignment. Only 5‘ and 3‘ of alignment has been briefly showed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0002: Alignment of amplified cDNA for canonical isoform of human CD19 reference sequence in NCBI database. Comparing the 1701 bp amplified sequence with reference sequence for short isoform (variant 2) of human CD19 showed complete alignment. Only 5‘ and 3‘ of alignment has been briefly showed.
Mentions: Initial optimization using Taq DNA polymerase showed specific band in annealing temperature of 64°C and 1 mM concentration of MgCl2. Subsequent PCR reaction using Pfu DNA polymerase led to a single band with expected size (1701 bp) (Figure 1A). PCR product was ligated into pGEM-T Easy vector by TA-cloning method and then transformed to competent DH5α bacteria using heat shock method. Blue-white selection was used for screening white colonies containing recombinant plasmid. Among them, eight white colonies were subjected to colony-PCR with gene-specific primers and two of them showed the expected band (Figure 1B). Then, miniprep was prepared from single positive colony and double digested with KpnI and HindIII restriction enzymes. Because of existence of one restriction site for KpnI in CD19 gene at nucleotide position 603, pGEM-T Easy-CD19 construct was subjected to partial digestion by KpnI and complete digestion by HindIII and led to excision of insert with length of 1701 bp (Figure 1C). Alignment of sequencing result with reference sequence in NCBI database showed complete matching with CD19 mRNA isoform 2 except at nucleotide position 4 where C was replaced with G in order to improve the expression of gene (Figure 2).

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