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A Highly Efficient Escherichia coli-Based Chromosome Engineering System Adapted for Recombinogenic Targeting and Subcloning of BAC DNA

Mouse Cancer Genetics Program, National Cancer Institute–Frederick, Frederick, Maryland, 21702
Genomics (Impact Factor: 2.79). 05/2001; 73(1):56-65. DOI: 10.1006/geno.2000.6451
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ABSTRACT Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective λ prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978–5983). Importantly, the recombination is proficient with DNA homologies as short as 30–50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3′ end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.

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Available from: E-Chiang Lee, Mar 19, 2014
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    • "A loxP-neo r -loxP expression cassette was inserted within a nonconserved region of Col2a1 intron 2 (approximately 350 bp downstream of exon 2) via another recombineering method (Lee et al., 2001). The targeting vector was generated from the engineered BAC using gap repair (Lee et al., 2001) into a plasmid containing a diphtheria toxin cassette for negative selection in ES cells. Resulting Col2a1-mIIC targeting vector DNA was prepared using a Qiagen maxi-prep protocol, linearized by restriction digest with Asc I and purified by phenol/chloroform extraction. "
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    • "A loxP-neo r -loxP expression cassette was inserted within a nonconserved region of Col2a1 intron 2 (approximately 350 bp downstream of exon 2) via another recombineering method (Lee et al., 2001). The targeting vector was generated from the engineered BAC using gap repair (Lee et al., 2001) into a plasmid containing a diphtheria toxin cassette for negative selection in ES cells. Resulting Col2a1-mIIC targeting vector DNA was prepared using a Qiagen maxi-prep protocol, linearized by restriction digest with Asc I and purified by phenol/chloroform extraction. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This study describes a new mechanism controlling the production of alternatively-spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively-spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codons in exon 6 (type IIC). This transcript is produced by utilization of another 5’ splice site present in exon 2. To determine the role of this IIC splicing event in vivo, we generated transgenic mice containing silent knock-in mutations at the IIC 5’ splice site (Col2a1-mIIC), thereby inhibiting production of IIC transcripts. Heterozygous and homozygous knock-in mice were viable and display no overt skeletal phenotype to date. However, RNA expression profiles revealed that chondrocytes in cartilage from an age range of Col2a1-mIIC mice produced higher levels of IIA and IID mRNAs and decreased levels of IIB mRNAs throughout pre-natal and post-natal development, when compared to chondrocytes from littermate control mice. Immunofluorescence analyses showed a clear increase in expression of embryonic type II collagen protein isoforms (i.e. containing the exon 2-encoded cysteine-rich (CR) protein domain) in cartilage extracellular matrix (ECM). Interestingly, at P14, P28 and P56, expression of embryonic Col2a1 isoforms in Col2a1-mIIC mice persisted in the pericellular domain of the ECM in articular and growth plate cartilage. We also show that persistent expression of the exon 2-encoded CR domain in the ECM of post-natal cartilage tissue may be due, in part, to the embryonic form of type XI collagen (the α3 chain of which is also encoded by the Col2a1 gene). In conclusion, expression of the Col2a1 IIC splice form may have a regulatory function in controlling alternative splicing of exon 2 to generate defined proportions of IIA, IID and IIB procollagen isoforms during cartilage development. Future studies will involve ultrastructural and biomechanical analysis of the collagen ECM to determine the effects of persistent mis-expression of embryonic collagen isoforms in mature cartilage tissue.
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