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Use of a promoter-trap retrovirus to identify and isolate genes involved in
differentiation of a myeloid progenitor cell line in vitro
JI Jonsson, Q Wu, K Nilsson and RA Phillips
Division of Immunology and Cancer Research, Hospital for Sick Children,
Toronto, Ontario, Canada.
Studies of gene regulation during early hematopoiesis and of the regulatory
network that controls differentiation and lineage commitment are hampered
by difficulties in isolating and growing stem cells and early progenitor
cells. These difficulties preclude the application of standard molecular
genetic approaches to these problems. As an alternative approach we have
introduced a lacZ-containing promoter-trap retrovirus into hematopoietic
cells. We used the interleukin-3- dependent mouse myeloid progenitor cell
32D as a model to identify transcriptionally active genes. The frequency of
integrations that led to transcription of the lacZ gene was estimated to be
0.5% of all integrations, of which 14% were downregulated on
differentiation of 32D cells towards neutrophils. Thus, one in every 1,000
to 2,000 integrations identified a developmentally regulated gene. Cellular
DNA sequences upstream of proviral integrations were isolated by inverse
polymerase chain reaction. Five were further characterized and we confirmed
by RNA expression analysis that they were downregulated on differentiation.
Sequence analysis revealed identification of novel genes with sequence
similarity to known genes. Considering the high efficiency of retroviral
infection, our study shows the feasibility of using promoter-trap vectors
to identity and isolate developmentally regulated genes from early
hematopoietic progenitors.
Volume 87,
Issue 5,
pp. 1771-1779,
03/01/1996
Copyright © 1996 by The American Society of Hematology
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