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Applications of a retroviral integrase towards substrate DNA in vivo Heale, John-Paule
Abstract
This study explored the efficacy of using retroviral integrase expressed transiently in vivo to mediate the recombination of exogenous DNA into host cell chromatin. Stable recombination of a substrate DNA into the genome of Baby Hamster Kidney (BHK) cells was achieved by co-transfecting them with a circular plasmid encoding Rous Sarcoma Virus (RSV) integrase (pIN) and a linearized plasmid (pNR) serving as a substrate for the integrase. Ndel linearized pNR (substrate DNA) mimicked the wild type RSV viral DNA in being a linear, double-stranded DNA molecule possessing terminal sequences recognizable by the integrase. Either a dihydrofolate reductase or neomycin cassette engineered into pNR served as a marker for recombination by conferring resistance to methotrexate or G418 selection, respectively. After 10 days growth in media supplemented with either 500µM methotrexate or 575µM G418, a small number of discrete colonies had formed on control plates containing cells which had been transfected only with substrate DNA. However, plates containing cells which had been co-transfected with both substrate DNA and pIN showed a ten-fold or higher increase in colony numbers over control plates. Sequence analysis of co-transfected BHK genomes identified many clonal recombinants; however none was a conserved, full length substrate DNA molecule expected from RSV IN mediated integration. Southern blot analysis of genomic DNA from co-transfected cells indicated that multiple copies of pNR had recombined with the BHK. Hence, in vivo, integrase increases the frequency of recombination of a recognizable substrate DNA molecule. However RSV IN mediated integration of substrate DNA was not observed.
Item Metadata
Title |
Applications of a retroviral integrase towards substrate DNA in vivo
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1997
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Description |
This study explored the efficacy of using retroviral integrase expressed transiently in vivo
to mediate the recombination of exogenous DNA into host cell chromatin. Stable recombination
of a substrate DNA into the genome of Baby Hamster Kidney (BHK) cells was achieved by
co-transfecting them with a circular plasmid encoding Rous Sarcoma Virus (RSV) integrase
(pIN) and a linearized plasmid (pNR) serving as a substrate for the integrase. Ndel linearized
pNR (substrate DNA) mimicked the wild type RSV viral DNA in being a linear,
double-stranded DNA molecule possessing terminal sequences recognizable by the integrase.
Either a dihydrofolate reductase or neomycin cassette engineered into pNR served as a marker for
recombination by conferring resistance to methotrexate or G418 selection, respectively. After 10
days growth in media supplemented with either 500µM methotrexate or 575µM G418, a small
number of discrete colonies had formed on control plates containing cells which had been
transfected only with substrate DNA. However, plates containing cells which had been
co-transfected with both substrate DNA and pIN showed a ten-fold or higher increase in colony
numbers over control plates. Sequence analysis of co-transfected BHK genomes identified many
clonal recombinants; however none was a conserved, full length substrate DNA molecule
expected from RSV IN mediated integration. Southern blot analysis of genomic DNA from
co-transfected cells indicated that multiple copies of pNR had recombined with the BHK. Hence,
in vivo, integrase increases the frequency of recombination of a recognizable substrate DNA
molecule. However RSV IN mediated integration of substrate DNA was not observed.
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Extent |
11925911 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-06-02
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0088791
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1997-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.