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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3349-3357
Improved Expression in Hematopoietic and Lymphoid Cells in Mice After
Transplantation of Bone Marrow Transduced With a Modified Retroviral
Vector
Stephanie Halene,
Lijun Wang,
Robert M. Cooper,
David C. Bockstoce,
Paul B. Robbins, and
Donald B. Kohn
From the Division of Research Immunology/Bone Marrow Transplantation,
Childrens Hospital Los Angeles, Los Angeles, CA; and the Department of
Pediatrics and Microbiology, University of Southern California School
of Medicine, Los Angeles, CA.
Retroviral vectors based on the Moloney murine leukemia virus
(MoMuLV) are currently the most commonly used vehicles for stable gene
transfer into mammalian hematopoietic cells. But, even with reasonable
transduction efficiency, expression only occurs in a low percentage of
transduced cells and decreases to undetectable levels over time. We
have previously reported the modified MND LTR
(myeloproliferative sarcoma virus enhancer,
negative control region deleted, dl587rev
primer-binding site substituted) to show increased expression frequency
and decreased methylation in transduced murine embryonic stem cells and
hematopoietic stem cells. We have now compared expression of the
enhanced green fluorescent protein (eGFP) from a vector using the
MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in
mature hematopoietic and lymphoid cells in the mouse bone marrow
transplant (BMT) model. In primary BMT recipients, we observed a higher
frequency of expression from the MND LTR (20% to 80%) in
hematopoietic cells of all lineages in spleen, bone marrow, thymus, and
blood compared with expression from the MoMuLV LTR (5% to 10%).
Expression from the MND LTR reached 88% in thymic T lymphocytes and
54% in splenic B lymphocytes for up to 8 months after BMT. The mean
fluorescence intensity of the individual cells, indicating the amount
of protein synthesized, was 6- to 10-fold higher in cells expressing
MNDeGFPSN compared with cells expressing LeGFPSN. Transduction
efficiencies determined by DNA polymerase chain reaction of vector copy
number were comparable for the 2 vectors. Therefore, the MND vector
offers an improved vehicle for reliable gene expression in
hematopoietic cells.
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