Enhancing Gene Targeting In Mammalian Cells By The Transient Down-Regulation Of Dna Repair Pathways.

Overview

The insertion of exogenous DNA at a specific genomic locus by gene targeting is an inefficient process in vertebrate cells. This is primarily due to the low frequency of targeted integration and high frequency of non-targeted integration, or random integration. Random integration is mediated through the nonhomologous end-joining pathway (NHEJ) and gene targeting through homologous recombination. In this work, we investigate the effects of transiently down-regulating important proteins involved in these ...
See more details below
Other sellers (Paperback)
  • All (2) from $71.96   
  • New (2) from $71.96   
Sending request ...

More About This Book

Overview

The insertion of exogenous DNA at a specific genomic locus by gene targeting is an inefficient process in vertebrate cells. This is primarily due to the low frequency of targeted integration and high frequency of non-targeted integration, or random integration. Random integration is mediated through the nonhomologous end-joining pathway (NHEJ) and gene targeting through homologous recombination. In this work, we investigate the effects of transiently down-regulating important proteins involved in these double-strand break repair (DSB) pathways in order to alter the inherent ability of a cell to perform gene targeting. To accomplish this we utilize RNAi technology. Ku70 and Xrcc4 are key components of NHEJ and represent different processes within the pathway. Ku70 is involved in DSB recognition and recruitment of the remaining NHEJ factors to the break and Xrcc4 is involved with ligation of the break. In this work we demonstrate differential effects of transiently depleting proteins involved in these early (Ku70) and late (Xrcc4) steps of the NHEJ pathway on random integration and gene targeting. HR occurs primarily in the S and G2-phases of the cell cycle. This suggests that gene targeting would be more efficient if cells were at these phases when interacting with targeting vectors. Furthermore, replication stress resulting in stalled and collapsed replication forks has been shown to stimulate HR. Here we used double thymidine block to synchronize cells to S-phase and aphidicolin to transiently stall DNA replication during the time of targeting vector/genome interaction. We demonstrate that transiently stalling replication during S-phase increases gene targeting efficiency in normal and transiently BLM helicase deficient cells whereas gene targeting efficiency is inhibited in NHEJ deficient cells. We also report on an intra-pathway off-target effect that resulted in illegitimate knockdown of NHEJ proteins Ku70 and Ku86 when using complex siRNA pools to deplete Xrcc4. We further demonstrate that neither mRNA quantity nor polysome distribution of Ku70 or Ku86 mRNAs were affected by Xrcc4 siRNA treatment, suggesting repression of translation by a post-initiation mechanism.
Read More Show Less

Product Details

  • ISBN-13: 9781243797032
  • Publisher: BiblioLabsII
  • Publication date: 9/9/2011
  • Pages: 120
  • Product dimensions: 7.44 (w) x 9.69 (h) x 0.25 (d)

Customer Reviews

Be the first to write a review
( 0 )
Rating Distribution

5 Star

(0)

4 Star

(0)

3 Star

(0)

2 Star

(0)

1 Star

(0)

Your Rating:

Your Name: Create a Pen Name or

Barnes & Noble.com Review Rules

Our reader reviews allow you to share your comments on titles you liked, or didn't, with others. By submitting an online review, you are representing to Barnes & Noble.com that all information contained in your review is original and accurate in all respects, and that the submission of such content by you and the posting of such content by Barnes & Noble.com does not and will not violate the rights of any third party. Please follow the rules below to help ensure that your review can be posted.

Reviews by Our Customers Under the Age of 13

We highly value and respect everyone's opinion concerning the titles we offer. However, we cannot allow persons under the age of 13 to have accounts at BN.com or to post customer reviews. Please see our Terms of Use for more details.

What to exclude from your review:

Please do not write about reviews, commentary, or information posted on the product page. If you see any errors in the information on the product page, please send us an email.

Reviews should not contain any of the following:

  • - HTML tags, profanity, obscenities, vulgarities, or comments that defame anyone
  • - Time-sensitive information such as tour dates, signings, lectures, etc.
  • - Single-word reviews. Other people will read your review to discover why you liked or didn't like the title. Be descriptive.
  • - Comments focusing on the author or that may ruin the ending for others
  • - Phone numbers, addresses, URLs
  • - Pricing and availability information or alternative ordering information
  • - Advertisements or commercial solicitation

Reminder:

  • - By submitting a review, you grant to Barnes & Noble.com and its sublicensees the royalty-free, perpetual, irrevocable right and license to use the review in accordance with the Barnes & Noble.com Terms of Use.
  • - Barnes & Noble.com reserves the right not to post any review -- particularly those that do not follow the terms and conditions of these Rules. Barnes & Noble.com also reserves the right to remove any review at any time without notice.
  • - See Terms of Use for other conditions and disclaimers.
Search for Products You'd Like to Recommend

Recommend other products that relate to your review. Just search for them below and share!

Create a Pen Name

Your Pen Name is your unique identity on BN.com. It will appear on the reviews you write and other website activities. Your Pen Name cannot be edited, changed or deleted once submitted.

 
Your Pen Name can be any combination of alphanumeric characters (plus - and _), and must be at least two characters long.

Continue Anonymously

    If you find inappropriate content, please report it to Barnes & Noble
    Why is this product inappropriate?
    Comments (optional)