Applications of the polymerase chain reaction in retroviral-mediated gene transfer of a foreign gene into hematopoietic cells of dogs and cats

Author

Glenn P. Niemeyer

Date of Award

8-1992

Degree Type

Thesis

Degree Name

Master of Science

Major

Comparative and Experimental Medicine

Major Professor

Clinton D. Lothrop Jr.

Committee Members

Albert Ichiki, Erby Wilkinson

Abstract

Gene transfer using retroviral vectors has become a widely used approach for introducing foreign genes into mammalian cells. Because of the efficiency of retroviral-mediated gene transfer, this system can be utilized to deliver therapeutically important genes into deficient cells as a treatment modality for certain genetic diseases of both man and animals. This study was undertaken to develop methods to analyze the many operational steps involved in retroviral-mediated transfer of foreign genes into hematopoietic progenitor cells of cats and dogs. Polymerase chain reaction protocols were developed which permitted us to detect, with a high degree of sensitivity and accuracy, retroviral transduced genes in experimental animals. In addition, the use of a retroviral vector expressing the cDNA for the human multidrug resistant gene (MDR1) as a dominant selectable marker was investigated. A Moloney murine leukemia virus based retroviral vector was used to transfer the bacterial neomycin resistance gene (neo^R) into canine and feline hematopoietic cells. Four cats and five dogs were lethally irradiated and reconstituted with autologous bone marrow that had been transduced with either the N2 or SAX retroviral vector. Bone marrow was obtained from gene transfer recipients at various times posttransplantation and tested for the expression of neo^R by the in vitro drug resistant colony forming unit granulocyte-macrophage assay. These results were corroborated utilizing the PGR method on gene transfer recipient bone marrow DNA. Experimental results showed expression and presence of retroviral vector sequences in all four cats and two dogs 30 to 48 day post-transplantation. Further sequential evaluation of feline bone marrow cells showed a low level of drug-resistant colony formation as well as the presence of vector sequences for greater than 200 days. Sequential expression and PGR analysis of canine bone marrow samples proved negative after approximately 80 days. A retroviral vector containing a full-length human MDR1 cDNA was used to evaluate the potential of in vivo selection in gene transfer protocols. Bone marrow cells from two dogs were transduced with the MDR1 retroviral vector. These animals were then administered myelosuppressive drugs at various time points post-transplantation. The number of drug resistant colony forming units increased five-fold in one animal during in vivo selection but decreased substantially once drug administration was stopped. PCR primers specific for human MDR1 cDNA were developed. These primers do not amplify canine or feline MDR genes. PCR analysis of bone marrow DNA from the two human MDR1 cDNA recipient dogs failed to detect the desired MDR1 sequences at multiple time points post-transplantation. Technical difficulties associated with the MDR1 retroviral vector used in this study, namely inefficient packaging and the generation of replication-competent virus, precluded additional studies with this vector. A new MDR1 expressing vector was constructed, and this vector (LMDR1) along with the PCR methodologies developed in this study should prove valuable in future experiments.

Recommended Citation

Niemeyer, Glenn P., "Applications of the polymerase chain reaction in retroviral-mediated gene transfer of a foreign gene into hematopoietic cells of dogs and cats. " Master's Thesis, University of Tennessee, 1992.
https://trace.tennessee.edu/utk_gradthes/12231