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Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts

Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2004; 32(16):e128. DOI: 10.1093/nar/gnh128
Source: PubMed

ABSTRACT The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes.

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    • "TAP -tagging mammalian proteomes is less straightforward for a number of reasons (Gingras et al., 2005 ) and are therefore less well represented. The combination of recombineering and tetraploid blastocyst complementation provides a means for large -scale TAP -tagging of mammalian genes and has been performed in mice (Zhou et al., 2004 ; Snow et al., 2008 ) and human cell lines (Bouwmeester et al., 2004 ; Brajenovic et al., 2004 ), and protocols exists to TAP -tag many mammalian proteins (Gingras et al., 2005 ). "
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    • "PSD-95 is a scaffold protein with three PDZ domains, an SH3 and a guanylate kinase domain that mediate protein interactions (Figure 1A). As mouse PSD-95 is known to have multiple isoforms generated by multiple promoters and all forms utilize a common C-terminus (Bence et al, 2005), the TAP tag was inserted into the open reading frame in the 3 0 -end before the stop codon of exon 19, using Escherichia coli recombineeringbased methods (Zhou et al, 2004) (Figure 1B). The final targeting vector, containing a 5 0 -end homology arm of 6.3 kb and a 3 0 -end homology arm of 2.9 kb, was transfected into ES cells and integration was detected using standard methods. "
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    • "Another exciting possibility is to knockin TAP-tags in mice: in this respect, a method was recently described for the rapid TAP-tagging of endogenous mouse genes. The speed of the technique arises from the use of recombineering and gap-repair rescue, which allow for the generation of mature transgenic mice within 3 months (Zhou et al. 2004 "
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    ABSTRACT: Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein-protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)-based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal-to-noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented.
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