Abstract
Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep+ hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep+, rep- (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep+ hybrid amplicon vector than with rep- or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep+ hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep- or standard amplicon vectors resulted in random integrations of the entire rep- hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1691-1702 |
| Number of pages | 12 |
| Journal | Gene Therapy |
| Volume | 10 |
| Issue number | 19 |
| DOIs | |
| State | Published - Sep 2003 |
| Externally published | Yes |
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Keywords
- AAV
- AAVS1
- Amplicon
- Transgenic mice
ASJC Scopus subject areas
- Genetics
Cite this
Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep+ hybrid amplicon vector. / Bakowska, J. C.; Di Maria, M. V.; Camp, S. M.; Wang, Y.; Allen, P. D.; Breakefield, X. O.
In: Gene Therapy, Vol. 10, No. 19, 09.2003, p. 1691-1702.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep+ hybrid amplicon vector
AU - Bakowska, J. C.
AU - Di Maria, M. V.
AU - Camp, S. M.
AU - Wang, Y.
AU - Allen, P. D.
AU - Breakefield, X. O.
PY - 2003/9
Y1 - 2003/9
N2 - Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep+ hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep+, rep- (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep+ hybrid amplicon vector than with rep- or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep+ hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep- or standard amplicon vectors resulted in random integrations of the entire rep- hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.
AB - Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep+ hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep+, rep- (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep+ hybrid amplicon vector than with rep- or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep+ hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep- or standard amplicon vectors resulted in random integrations of the entire rep- hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.
KW - AAV
KW - AAVS1
KW - Amplicon
KW - Transgenic mice
UR - http://www.scopus.com/inward/record.url?scp=0041911481&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0041911481&partnerID=8YFLogxK
U2 - 10.1038/sj.gt.3302061
DO - 10.1038/sj.gt.3302061
M3 - Article
VL - 10
SP - 1691
EP - 1702
JO - Gene Therapy
JF - Gene Therapy
SN - 0969-7128
IS - 19
ER -