CD34 splice variant: an attractive marker for selection of gene-modified cells
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CD34 splice variant: an attractive marker for selection of gene-modified cells. / Fehse, B; Richters, A; Putimtseva-Scharf, K; Klump, H; Li, Z; Ostertag, W; Zander, A R; Baum, Christopher.
in: MOL THER, Jahrgang 1, Nr. 5 Pt 1, 05.2000, S. 448-456.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - CD34 splice variant: an attractive marker for selection of gene-modified cells
AU - Fehse, B
AU - Richters, A
AU - Putimtseva-Scharf, K
AU - Klump, H
AU - Li, Z
AU - Ostertag, W
AU - Zander, A R
AU - Baum, Christopher
PY - 2000/5
Y1 - 2000/5
N2 - This study presents a promising selection system for gene-modified cells other than human hematopoietic progenitor and endothelial cells based on transgenic expression of human CD34. Three retrovirally transduced variants of CD34 were compared, differing in the length of their cytoplasmic domains. These were the full-length transmembrane protein (flCD34), a truncated form (tCD34) that is found as a naturally occurring splice variant and has a partial deletion of the cytoplasmic domain for signal transduction, and an engineered variant which is completely deprived of its cytoplasmic tail (dCD34). All three variants allowed selection of gene-modified cells using commercially available immunoaffinity technology. However, examination by flow cytometry as well as by Southern, Northern, and Western blot revealed that dCD34, as opposed to tCD34, is not stably anchored in the membrane and thus is expressed at low levels on the surface of transduced cells. Therefore, tCD34 was chosen as the more promising candidate for a clinically applicable cell surface marker. We show that gene-modified human primary T lymphocytes expressing tCD34 can be enriched to high purity (>95%) using clinically approved immunoaffinity columns. In addition, we demonstrate the utility of tCD34 for surface marking of murine hematopoietic cells in vivo, including primary T lymphocytes detected 9 weeks after bone marrow transplantation.
AB - This study presents a promising selection system for gene-modified cells other than human hematopoietic progenitor and endothelial cells based on transgenic expression of human CD34. Three retrovirally transduced variants of CD34 were compared, differing in the length of their cytoplasmic domains. These were the full-length transmembrane protein (flCD34), a truncated form (tCD34) that is found as a naturally occurring splice variant and has a partial deletion of the cytoplasmic domain for signal transduction, and an engineered variant which is completely deprived of its cytoplasmic tail (dCD34). All three variants allowed selection of gene-modified cells using commercially available immunoaffinity technology. However, examination by flow cytometry as well as by Southern, Northern, and Western blot revealed that dCD34, as opposed to tCD34, is not stably anchored in the membrane and thus is expressed at low levels on the surface of transduced cells. Therefore, tCD34 was chosen as the more promising candidate for a clinically applicable cell surface marker. We show that gene-modified human primary T lymphocytes expressing tCD34 can be enriched to high purity (>95%) using clinically approved immunoaffinity columns. In addition, we demonstrate the utility of tCD34 for surface marking of murine hematopoietic cells in vivo, including primary T lymphocytes detected 9 weeks after bone marrow transplantation.
KW - Animals
KW - Antigens, CD34/biosynthesis
KW - Antigens, Surface/biosynthesis
KW - Blotting, Western
KW - Bone Marrow/metabolism
KW - Cell Separation/methods
KW - DNA/analysis
KW - DNA Primers/chemistry
KW - DNA, Recombinant
KW - Flow Cytometry
KW - Gene Expression
KW - Genetic Vectors
KW - Humans
KW - Immunomagnetic Separation
KW - Jurkat Cells
KW - Mice
KW - Mice, Inbred C57BL
KW - Nucleotide Mapping
KW - Retroviridae/genetics
KW - T-Lymphocytes/cytology
KW - Transfection
U2 - 10.1006/mthe.2000.0068
DO - 10.1006/mthe.2000.0068
M3 - SCORING: Journal article
C2 - 10933966
VL - 1
SP - 448
EP - 456
JO - MOL THER
JF - MOL THER
SN - 1525-0016
IS - 5 Pt 1
ER -