Abstract:
:In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to as delitto perfetto, that combines the versatility of synthetic oligonucleotides for targeting with the practicality of a general selection system. It provides for an enormously wide variety of genome modifications via homologous recombination. Exceptional high frequencies of mutations are reached when a site-specific double-strand break (DSB) is induced within the locus targeted by the synthetic oligonucleotides. Presented in this chapter is an in-depth description of a series of applications of the delitto perfetto strategy for mutagenesis and chromosome modification both with and without the induction of a DSB, along with the procedures and materials.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Storici F,Resnick MAdoi
10.1016/S0076-6879(05)09019-1subject
Has Abstractpub_date
2006-01-01 00:00:00pages
329-45eissn
0076-6879issn
1557-7988pii
S0076-6879(05)09019-1journal_volume
409pub_type
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abstract::Mdm2 is a negative regulator of p53 activity and functions as an E3 ubiquitin ligase of p53. Inhibition of mdm2 E3 ligase activity will block ubiquitination and subsequent proteasome-mediated degradation of p53, resulting in the stabilization of p53 protein that could lead to the restoration of its tumor-suppressor ac...
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