Molecular Cloning And Expression Of CDNA For Human Interleukin-3 In Mammalian Cells

Interleukin-3(IL-3), also called multi-CSF, is one of the lymphokines, produced by activated T cells. IL-3 is known to be important in the regulation of heamopoiesis. Its dysregulation may be related to tumorigenesis. In the present paper, molecular cloning , sequence analysis and retroviral mediated gene transfer of human IL-3 to mammalian cells is presented.The lymphocytes separated from human tonsils were cultured in regular medium supplemented with PHA-P and TPA for 24 hours . Total RNA was isolated by using guanidium isothiocyanate/CsCI method. Reverse trascription for synthesizing the first-strand cDNA as the template for PCR. Two 27-base oligonucleotide primers were made on a AB1 DNA synthesizer. They contained an artificial restriction site (BamHI, HindIII) at the 5' endof each one. The synthesized primers were purified through "OPC" column. Using Cetus gene amplification system, PCR was performed for 25 cycles in a total volume of 100ul, 200 picomoles of each primer, 200 micromoles of dNTP, 2.5 units of Taq DNA polymerase. After PCR reaction, The amplified human IL-3 cDNA fragment was purified on a 1.5% agarose gel and recovered by DEAE paper, The purified hIL-3 was undirectionally cloned into M13mp18 or M13mp19, and sequenced in both directions, Sequence analysis data...