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Retroviral vector design for long-term expression in murine hematopoietic
cells in vivo
PH Correll, S Colilla and S Karlsson
Molecular and Medical Genetics Section, National Institute of Neurological
Disease and Stroke, National Institutes of Health, Bethesda, MD.
A series of retroviral vectors containing the human glucocerebrosidase (GC)
cDNA driven by various promoters have been constructed in an attempt to
discover which vector design can most efficiently transduce murine
hematopoietic stem cells (HSCs) and drive expression of the transferred
gene in hematopoietic cells of mice reconstituted with the transduced stem
cells. The simplest vector, LG, in which the GC gene is driven by the viral
LTR, was the most efficient vector at infecting HSCs, with an average viral
copy number in hematopoietic tissues of 3 copies/cell in recipient mice. In
general, the viral vectors that contained any additional promoters or
enhancers to drive expression of either the GC gene or a selectable marker
gene (Neo) had lower titers and/or transduced HSCs at a lower efficiency.
This was seen most markedly when the human phosphoglycerate (PGK) promoter
was used to drive the human GC cDNA. Despite repeated attempts to obtain a
high titer producer clone, this virus consistently produced low titers and
subsequently resulted in the lowest proviral copy numbers in long-term
reconstituted mice. Only the viral LTR and PGK promoter were capable of
driving significant levels of human GC RNA in hematopoietic cells of
long-term reconstituted mice, with a much lower level of RNA generated by
an internal herpes TK or SV40 immediate early promoter. Insertion of the
internal transcription unit in the opposite orientation relative to the
viral LTRs had a detrimental effect on gene expression. The levels of RNA
generated by a hybrid LTR containing the myeloproliferative sarcoma virus
enhancer were higher in bone marrow-derived macrophages than in nonadherent
cells of the bone marrow when compared with the LG vector. The presence of
an internal promoter to drive expression of the human GC cDNA did not seem
to have a detrimental effect on expression levels from the viral LTR. In
fact, in the presence of an internal TK or PGK promoter expression from the
LTR was increased despite the presence of lower proviral copy numbers.
Insertion of a second gene (Neo) into the vector had a negative impact on
long-term expression in hematopoietic cells in vivo; however, this seems to
be due solely to the lower transduction efficiency of this vector. Overall,
the highest levels of GC activity in macrophages of long-term reconstituted
mice were generated by the LG vector; however, these levels were
variable.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 84,
Issue 6,
pp. 1812-1822,
09/15/1994
Copyright © 1994 by The American Society of Hematology
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