A sensitive technique to monitor gene transfer and expression in bone marrow stem cells.
The polymerase chain reaction technique (PCR), a primer-mediated enzymatic amplification of specific target sequences, was used to monitor gene transfer into hematopoietic progenitor stem cells. A gene coding for human interleukin 1 alpha (IL-1 alpha) was cotransfected with the rous sarcoma virus (RSV) CAT plasmid into mouse bone marrow cells by electroporation. Individual chloramphenicol (CAM)-resistant bone marrow progenitor colonies (granulocyte-macrophage colony-forming units; CFU-GM) containing 50-100 cells were analyzed by PCR for the presence and expression of IL-1 alpha and CAT sequences. Amplified IL-1 alpha DNA sequences were detected from a 50-cell CFU-GM colony. CAT and IL-1 alpha RNA expression was demonstrated from the CAM-resistant CFU-GM colonies.
Narayanan
R
R
Department of Molecular Genetics, Hoffmann-La Roche Incorporated, Nutley, New Jersey 07110.
Tare
N S
NS
Benjamin
W R
WR
Gubler
U
U
D000818:Animals; D001853:Bone Marrow / Q000502:physiology*; D001854:Bone Marrow Cells; D002999:Clone Cells; D003115:Colony-Stimulating Factors / Q000494:pharmacology; D005784:Gene Amplification; D005786:Gene Expression Regulation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D007375:Interleukin-1 / Q000235:genetics*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D003115:Colony-Stimulating Factors; D006133:Growth Substances; D007375:Interleukin-1; D012333:RNA, Messenger; D016178:Granulocyte-Macrophage Colony-Stimulating Factor