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An Overview on the Generation of BAC Transgenic Mice for Neuroscience Research

X. William Yang¹, Shiaoching Gong²

¹Department of Psychiatry and Biobehavioral Sciences, Center for Neurobehavioral Genetics, Neuropsychiatric Institute, David Geffen School of Medicine at UCLA, Los Angeles, California
²Rockefeller University, New York, New York

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 5.20

DOI:

10.1002/0471142301.ns0520s31

Online Posting Date:

May, 2005

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Abstract

This unit provides a comprehensive overview on the generation of transgenic mice using bacterial artificial chromosomes (BACs), and the application of BAC transgenic mice in neuroscience research. In the first section, advantages of the BAC transgenic approach compared to the conventional transgenic approach are summarized. In the second section, important considerations in designing BAC transgenic constructs are outlined. Four commonly used BAC transgenic construct designs are also outlined. Concepts of modifying BACs by homologous recombination in E. coli to introduce a variety of mutations into BACs, and important steps to characterize a modified BAC prior to the generation of transgenic mice are also presented. In the final section, some of the important applications of BAC transgenic mice in neuroscience research, including studying gene expression, gene function, mapping neuronal circuitry, and modeling human diseases, are described.

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Unit Introduction
Transgenic Mice: Some General Considerations
BAC Transgenic Construct Design
BAC Modification by Homologous Recombination in E. coli
Characterization of Modified BACs and Preparation of BAC DNA for Microinjections
Mouse Strain Considerations
Applications of BAC Transgenic Mice in Neuroscience Research
Literature Cited
Figures

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Figures

Figure 5.20.1

Four basic designs of BAC transgenic constructs. Construct 1 (CS-1) and construct 2 (CS-2) are designed to overexpress a transgenic open reading frame (Tg-ORF), such as the green fluorescent protein, lacZ, Cre, and others, but the driver gene itself is not overexpressed. Both CS-1 and CS-2 have Tg-ORF inserted in front of the translation initiation sequence (ATG) of the driver gene. The difference between these two constructs is that CS-1 has Tg-ORF inserted into exon 1 and uses an endogenous polyA signal from the driver gene, and Tg-ORF2 is not inserted into exon 1 and uses an exogenous polyA signal (PA). Construct 3 (CS-3) uses an internal ribosomal entry sequence (IRES) to drive overexpression of both the driver gene and a Tg-ORF. The IRES.Tg-ORF is often inserted into the 3¢-untranslated region (3¢-UTR) of the driver gene. Finally, construct-4 (CS-4) is designed to use IRES to overexpress two transgenes of interest, but the driver gene is not overexpressed.

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Figure 5.20.2

Modification of BAC by homologous recombination in E. coli. A shuttle vector (pLD53.SCAB) is used for modification. This vector contains an R6K origin of replication, a RecA gene to support recombination, a SacB gene for negative selection, and a recombination cassette containing two small homology boxes A and B (~500-bp each) flanking a modification to be introduced (e.g., EGFP gene). The shuttle vector plasmid is electroporated into the BAC host bacteria. Some of the shuttle vectors can undergo homologous recombination with the BAC through either the A or B boxes, resulting in a complete integration of the shuttle vector into the BAC to form a co-integrate BAC. Bacteria containing co-integrate BACs can be selected by growth on chloramphenicol (chlor) and ampicillin (Amp). A small percentage of bacteria with co-integrates can undergo a second homologous recombination step through the A or B boxes (called resolution), resulting in the excision of the shuttle vector. The excised shuttle vector is automatically lost, and the bacteria with resolved BAC can be selected by growth on chloramphenicol and sucrose (sucrose selects for loss of the SacB gene). If co-integration and resolution occur through different homology boxes, the result is a correctly modified BAC with precise placement of the EGFP marker gene on a chosen locus on the BAC.

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Literature Cited

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