Unit

UNIT 23.14 Recombineering-Based Procedure for Creating BAC Transgene Constructs for Animals and Cell Lines

  1. Steven M. Hollenback,
  2. Suzanne Lyman,
  3. JrGang Cheng

Published Online: 1 JUL 2011

DOI: 10.1002/0471142727.mb2314s95

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Hollenback, S. M., Lyman, S. and Cheng, J. 2011. Recombineering-Based Procedure for Creating BAC Transgene Constructs for Animals and Cell Lines. Current Protocols in Molecular Biology. 95:23.14:23.14.1–23.14.28.

Author Information

  1. Neuroscience Center, UNC-Chapel Hill, Chapel Hill, North Carolina

Publication History

  1. Published Online: 1 JUL 2011
  2. Published Print: JUL 2011

Abstract

The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large-scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue-specific expression as a knock-in of the same gene with less effort and shorter time of establishment. The λ-RED recombinogenic system has been used to manipulate DNA constructs with site-directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used here to modify many BACs with various transgenes. The recombineering plasmid, pKD46, is used to fabricate BAC transgenic constructs that can be used in generating transgenic organisms as well as used in mammalian cell culture. Curr. Protoc. Mol. Biol. 95:23.14.1-23.14.28. © 2011 by John Wiley & Sons, Inc.

Keywords:

  • BAC;
  • recombineering;
  • transgene