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Conventional gene targeting protocols lead to loss of targeted cells when applied to a silent gene locus in primary fibroblasts

Paper ID Volume ID Publish Year Pages File Format Full-Text
25743 43596 2006 9 PDF Available
Title
Conventional gene targeting protocols lead to loss of targeted cells when applied to a silent gene locus in primary fibroblasts
Abstract

Gene targeting in livestock fibroblasts has proven difficult to achieve, particularly if the target gene is silent. We first tested whether efficient gene targeting at the transcriptionally active ovine α1(I) procollagen (COL1A1) locus required the use of a promoter trap vector. We compared gene targeting frequencies at the ovine COL1A1 locus using both a promoter trap and a non-promoter trap selection strategy. We demonstrated that targeted cells could be isolated regardless of whether an enrichment step (promoter trap) was used. Next, we used our optimised protocol to target a non-expressed gene, ovine β-casein. We obtained clones that were scored positive by PCR for the targeting event, but were negative after cell expansion and Southern analysis. We propose that targeted cells were initially generated but that they were at a selective growth disadvantage during culture. We suggest modifications to the conventional targeting protocol that would prevent such loss of targeted cells.

Keywords
Gene targeting; Promoter trap; Transcriptionally silent genes; Beta-casein; COL1A1; Sheep primary fibroblasts
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Conventional gene targeting protocols lead to loss of targeted cells when applied to a silent gene locus in primary fibroblasts
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 125, Issue 2, 1 September 2006, Pages 185–193
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us