Direct display of hematopoietic tyrosine kinase receptor expression profiles in KG1 cells by PCR using degenerate primers.
Hematopoietic tyrosine kinase receptors (HGF-TKRs or class III TKRs) are essential for the growth and differentiation of hematopoietic cells. In this report we present a novel method that generates expression profiles of these receptors. The method was tested and optimized using the myeloblastic/ promyelocytic cell line KG1. The method involves PCR of cDNA using class III-specific degenerate primers and subsequent restriction enzyme digests of the 147 bp amplicons followed by fractionation on denaturing poly-acrylamide gels. This primary fingerprint of KG1 revealed equal expression of c-kit and flt3 and to a lesser extent PDGF-R alpha and c-fms. One residual band of unknown origin was seen and appeared to be the proto-oncogene RET following cloning and sequence analysis. This tyrosine kinase receptor is known to play an important role in neural development. In order to detect less abundantly expressed sequences, a secondary fingerprint was generated by pre-digestion of the receptors present in the primary expression profile and subsequent amplification of the residual band. No other tyrosine kinase receptors were observed in KG1. In conclusion, this method allows direct visualization of expression of the HGF-TKRs and has the potential to detect novel homologous receptors.
Visser
M
M
Laboratory for Experimental Hematology, Department of Hematology, Leiden University Hospital, The Netherlands.
Sonneveld
R D
RD
Willemze
R
R
Landegent
J E
JE
D001483:Base Sequence; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D016172:DNA Fingerprinting; D017931:DNA Primers; D029721:Drosophila Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007938:Leukemia; D008168:Lung; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000096:biosynthesis; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000096:biosynthesis; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis / Q000096:biosynthesis*; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000032:analysis / Q000096:biosynthesis; D020796:Receptor, Platelet-Derived Growth Factor alpha; D017479:Receptors, Platelet-Derived Growth Factor / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D051941:fms-Like Tyrosine Kinase 3
D017931:DNA Primers; D029721:Drosophila Proteins; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; C497970:FLT3 protein, human; D019009:Proto-Oncogene Proteins c-kit; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor; D020796:Receptor, Platelet-Derived Growth Factor alpha; D017479:Receptors, Platelet-Derived Growth Factor; C416649:Ret protein, Drosophila; D051941:fms-Like Tyrosine Kinase 3