Identification of genes induced by factor deprivation in hematopoietic cells undergoing apoptosis using gene-trap mutagenesis and site-specific recombination.
A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/loxP) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus-thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.
Russ
A P
AP
Department of Hematology, University of Frankfurt Medical School, Germany.
Friedel
C
C
Ballas
K
K
Kalina
U
U
Zahn
D
D
Strebhardt
K
K
von Melchner
H
H
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019426:Integrases / Q000096:biosynthesis / Q000235:genetics; D007377:Interleukin-3 / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D019868:Kanamycin Kinase; D007700:Kinetics; D051379:Mice; D016296:Mutagenesis; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins
D017931:DNA Primers; D007377:Interleukin-3; D011994:Recombinant Proteins; D014764:Viral Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D013937:Thymidine Kinase; D019868:Kanamycin Kinase; C045073:Cre recombinase; D019426:Integrases