Cargando…

Improved methods for the generation of human gene knockout and knockin cell lines

Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not bee...

Descripción completa

Detalles Bibliográficos
Autores principales: Topaloglu, Ozlem, Hurley, Paula J., Yildirim, Ozlem, Civin, Curt I., Bunz, Fred
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255732/
https://www.ncbi.nlm.nih.gov/pubmed/16214806
http://dx.doi.org/10.1093/nar/gni160
_version_ 1782125790826070016
author Topaloglu, Ozlem
Hurley, Paula J.
Yildirim, Ozlem
Civin, Curt I.
Bunz, Fred
author_facet Topaloglu, Ozlem
Hurley, Paula J.
Yildirim, Ozlem
Civin, Curt I.
Bunz, Fred
author_sort Topaloglu, Ozlem
collection PubMed
description Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of β-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells.
format Text
id pubmed-1255732
institution National Center for Biotechnology Information
language English
publishDate 2005
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-12557322005-10-24 Improved methods for the generation of human gene knockout and knockin cell lines Topaloglu, Ozlem Hurley, Paula J. Yildirim, Ozlem Civin, Curt I. Bunz, Fred Nucleic Acids Res Methods Online Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of β-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells. Oxford University Press 2005 2005-10-07 /pmc/articles/PMC1255732/ /pubmed/16214806 http://dx.doi.org/10.1093/nar/gni160 Text en © The Author 2005. Published by Oxford University Press. All rights reserved
spellingShingle Methods Online
Topaloglu, Ozlem
Hurley, Paula J.
Yildirim, Ozlem
Civin, Curt I.
Bunz, Fred
Improved methods for the generation of human gene knockout and knockin cell lines
title Improved methods for the generation of human gene knockout and knockin cell lines
title_full Improved methods for the generation of human gene knockout and knockin cell lines
title_fullStr Improved methods for the generation of human gene knockout and knockin cell lines
title_full_unstemmed Improved methods for the generation of human gene knockout and knockin cell lines
title_short Improved methods for the generation of human gene knockout and knockin cell lines
title_sort improved methods for the generation of human gene knockout and knockin cell lines
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255732/
https://www.ncbi.nlm.nih.gov/pubmed/16214806
http://dx.doi.org/10.1093/nar/gni160
work_keys_str_mv AT topalogluozlem improvedmethodsforthegenerationofhumangeneknockoutandknockincelllines
AT hurleypaulaj improvedmethodsforthegenerationofhumangeneknockoutandknockincelllines
AT yildirimozlem improvedmethodsforthegenerationofhumangeneknockoutandknockincelllines
AT civincurti improvedmethodsforthegenerationofhumangeneknockoutandknockincelllines
AT bunzfred improvedmethodsforthegenerationofhumangeneknockoutandknockincelllines