Nucleic Acids Research, 2002, Vol. 30, No. 10 e41
© 2002 Oxford University Press
A reliable and efficient method for deleting operational sequences in PACs and BACs
Genetics Interdisciplinary Program and 1Departments of Internal Medicine and Physiology & Biophysics, 2191 Medical Laboratory, University of Iowa College of Medicine, Iowa City, IA 52242, USA
P1-derived artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have become very useful as tools to study gene expression and regulation in cells and in transgenic mice. They carry large fragments of genomic DNA (
100 kb) and therefore may contain all of the cis-regulatory elements required for expression of a gene. Because of this, even when inserted randomly in the genome, they can emulate the native environment of a gene resulting in a tightly regulated pattern of expression. Because these large genomic clones often contain DNA sequences which can manipulate chromatin at the local level, they become immune to position effects which affect expression of smaller transgenes, and thus their expression is proportional to copy number. Transgenic mice containing large BACs and PACs have become excellent models to examine the regulation of gene expression. Their usefulness would certainly be increased if easy and efficient methods are developed to manipulate them. We describe herein a method to make deletion mutations reliably and efficiently using a novel modification of the Chi-stimulated homologous recombination method. Specifically, we generated and employed a Lox511 ‘floxed’ CAM resistance marker that first affords selection for homologous recombination in Escherichia coli, and then can be easily deleted leaving only a single Lox511 site as the footprint.
* To whom correspondence should be addressed. Tel: +1 319 335 7604; Fax: +1 319 353 5350; Email: curt-sigmund{at}uiowa.edu
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  X. Zhou and C. D. Sigmund Chorionic enhancer is dispensable for regulated expression of the human renin gene Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2008; 294(2): R279 - R287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhoux, D. R. Davis, and C. D. Sigmund The Human Renin Kidney Enhancer Is Required to Maintain Base-line Renin Expression but Is Dispensable for Tissue-specific, Cell-specific, and Regulated Expression J. Biol. Chem., November 17, 2006; 281(46): 35296 - 35304. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Shakes, D. M. Garland, D. K. Srivastava, K. R. Harewood, and P. K. Chatterjee Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors Nucleic Acids Res., August 1, 2005; 33(13): e118 - e118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Nistala, X. Zhang, and C. D. Sigmund Differential expression of the closely linked KISS1, REN, and FLJ10761 genes in transgenic mice Physiol Genomics, March 12, 2004; 17(1): 4 - 10. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Bianco, H. L. Keen, J. L. Lavoie, and C. D. Sigmund Untraditional methods for targeting the kidney in transgenic mice Am J Physiol Renal Physiol, December 1, 2003; 285(6): F1027 - F1033. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Liu, X. Huang, and C. D. Sigmund Identification of a Nuclear Orphan Receptor (Ear2) as a Negative Regulator of Renin Gene Transcription Circ. Res., May 16, 2003; 92(9): 1033 - 1040. [Abstract] [Full Text] [PDF]
|
|