PMID- 24521444
OWN - NLM
STAT- MEDLINE
DCOM- 20140911
LR  - 20220129
IS  - 1471-2199 (Electronic)
IS  - 1471-2199 (Linking)
VI  - 15
DP  - 2014 Feb 13
TI  - Inverted terminal repeats of adeno-associated virus decrease random integration 
      of a gene targeting fragment in Saccharomyces cerevisiae.
PG  - 5
LID - 10.1186/1471-2199-15-5 [doi]
AB  - BACKGROUND: Homologous recombination mediated gene targeting is still too 
      inefficient to be applied extensively in genomics and gene therapy. Although 
      sequence-specific nucleases could greatly stimulate gene targeting efficiency, 
      the off-target cleavage sites of these nucleases highlighted the risk of this 
      strategy. Adeno-associated virus (AAV)-based vectors are used for specific gene 
      knockouts, since several studies indicate that these vectors are able to induce 
      site-specific genome alterations at high frequency. Since each targeted event is 
      accompanied by at least ten random integration events, increasing our knowledge 
      regarding the mechanisms behind these events is necessary in order to understand 
      the potential of AAV-mediated gene targeting for therapy application. Moreover, 
      the role of AAV regulatory proteins (Rep) and inverted terminal repeated 
      sequences (ITRs) in random and homologous integration is not completely known. In 
      this study, we used the yeast Saccharomyces cerevisiae as a genetic model system 
      to evaluate whether the presence of ITRs in the integrating plasmid has an effect 
      on gene targeting and random integration. RESULTS: We have shown that the 
      presence of ITRs flanking a gene targeting vector containing homology to its 
      genomic target decreased the frequency of random integration, leading to an 
      increase in the gene targeting/random integration ratio. On the other hand, the 
      expression of Rep proteins, which produce a nick in the ITR, significantly 
      increased non-homologous integration of a DNA fragment sharing no homology to the 
      genome, but had no effect on gene targeting or random integration when the DNA 
      fragment shared homology with the genome. Molecular analysis showed that ITRs are 
      frequently conserved in the random integrants, and that they induce 
      rearrangements. CONCLUSIONS: Our results indicate that ITRs may be a useful tool 
      for decreasing random integration, and consequently favor homologous gene 
      targeting.
FAU - Galli, Alvaro
AU  - Galli A
AD  - Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via 
      Moruzzi 1, 56125 Pisa, Italy. alvaro.galli@ifc.cnr.it.
FAU - Cervelli, Tiziana
AU  - Cervelli T
LA  - eng
GR  - GGP09166/TI_/Telethon/Italy
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20140213
PL  - England
TA  - BMC Mol Biol
JT  - BMC molecular biology
JID - 100966983
RN  - 0 (Viral Proteins)
SB  - IM
MH  - Dependovirus/*genetics
MH  - Gene Targeting/methods
MH  - Genetic Therapy/methods
MH  - Genetic Vectors/genetics
MH  - Recombination, Genetic/*genetics
MH  - Saccharomyces cerevisiae/*genetics/virology
MH  - Terminal Repeat Sequences/*genetics
MH  - Viral Proteins/genetics
MH  - Virus Integration/*genetics
PMC - PMC3925961
EDAT- 2014/02/14 06:00
MHDA- 2014/09/12 06:00
PMCR- 2014/02/13
CRDT- 2014/02/14 06:00
PHST- 2013/10/16 00:00 [received]
PHST- 2014/02/06 00:00 [accepted]
PHST- 2014/02/14 06:00 [entrez]
PHST- 2014/02/14 06:00 [pubmed]
PHST- 2014/09/12 06:00 [medline]
PHST- 2014/02/13 00:00 [pmc-release]
AID - 1471-2199-15-5 [pii]
AID - 10.1186/1471-2199-15-5 [doi]
PST - epublish
SO  - BMC Mol Biol. 2014 Feb 13;15:5. doi: 10.1186/1471-2199-15-5.