Farzaneh Shafaghat
1,2,3, Hajar Abbasi-Kenarsari
4, Jafar Majidi
4,5, Ali Akbar Movassaghpour
6, Dariush Shanehbandi
5, Tohid Kazemi
5*1 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
2 Department of Immunology, International Branch of Aras, Tabriz University of Medical Sciences, Tabriz, Iran.
3 Students' Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
4 Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
5 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
6 Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
Purpose: Transmembrane CD34 glycoprotein is the most
important marker for identification, isolation and enumeration of hematopoietic
stem cells (HSCs). We aimed in this study to clone the cDNA coding for human
CD34 from KG1a cell line and stably express in mouse fibroblast cell line
NIH-3T3. Such artificial cell line could be useful as proper immunogen for
production of mouse monoclonal antibodies.
Methods: CD34 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 TA-cloning 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.
Results: 1158 bp specific band was aligned completely to reference
sequence in NCBI database corresponding to long isoform of human CD34.
Transient and stable expression of human CD34 on transfected NIH-3T3 mouse
fibroblast cells was achieved (25% and 95%, respectively) as shown by flow
cytometry.
Conclusion: Cloning and stable expression of human
CD34 cDNA was successfully performed and validated by standard flow cytometric
analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3
cells could be useful as immunogen in production of diagnostic monoclonal
antibodies against human CD34. This approach could bypass the need for
purification of recombinant proteins produced in eukaryotic expression systems.