Veterinary Research Communications

, Volume 30, Issue 8, pp 881–901

Optimized Transduction of Canine Paediatric CD34+ Cells Using an MSCV-based Bicistronic Vector

  • S. E. Suter
  • T. A. Gouthro
  • P. A. McSweeney
  • R. A. Nash
  • M. E. Haskins
  • P. J. Felsburg
  • P. S. Henthorn
Article

DOI: 10.1007/s11259-006-3356-7

Cite this article as:
Suter, S.E., Gouthro, T.A., McSweeney, P.A. et al. Vet Res Commun (2006) 30: 881. doi:10.1007/s11259-006-3356-7

Abstract

We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (γc) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34+ bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting ∼75% of Cf2Th cells. An average of 11% of highly enriched canine CD34+ cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine γc cDNA produced a γc protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34+ BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

Keywords

XSCID CD34+ retroviral transduction amphotropic retrovirus gamma c 

Abbreviations

BM

bone marrow

BMT

bone marrow transplantation

EGFP

enhanced green fluorescent protein

γc

gamma c

HPC

haematopoietic progenitor cell

MoMLV

Moloney murine leukaemia virus

MSCV

murine stem cell virus

SRC

SCID repopulating cells

SP

side population

XSCID

X-linked severe combined immunodeficiency

Copyright information

© Springer Science + Business Media, Inc. 2006

Authors and Affiliations

  • S. E. Suter
    • 1
  • T. A. Gouthro
    • 1
  • P. A. McSweeney
    • 3
  • R. A. Nash
    • 4
  • M. E. Haskins
    • 2
  • P. J. Felsburg
    • 1
  • P. S. Henthorn
    • 1
  1. 1.Section of Medical GeneticsDepartment of Clinical StudiesPhiladelphiaPennsylvania
  2. 2.Laboratory of Pathology, Department of PathobiologyUniversity of Pennsylvania School of Veterinary MedicinePhiladelphiaPennsylvania
  3. 3.Bone Marrow Transplant ProgramUniversity of ColoradoDenverColorado
  4. 4.Fred Hutchinson Cancer Research CenterSeattleUSA