BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals - PubMed (original) (raw)

Tagging cassettes and 96-well recombineering pipeline. (a,b) Tagging cassettes for C- and N-terminal end tagging. Schematic representation of a genomic region that contains the gene of interest (orange) and the cassettes for tagging at the N or C end. (a) In the N-terminal cassette, the neomycin-kanamycin resistance gene (neo) is placed inside an artificial intron flanked by loxP sites. (b) The C-terminal tag is inserted as a cassette with a neomycin resistance gene downstream of an internal ribosome entry site (IRES:neo). EGFP, enhanced green fluorescent protein; gb2, gb3, bacterial promoters; P, PreScission cleavage site; pgk, phosphoglycerate kinase (PGK) promoter; S, S-peptide; sa, splice acceptor; sd, splice donor; T, TEV cleavage site. (c) 96-well-format BAC recombineering, with actual time required for each experimental step shown. The cartoons at right represent the events that occur at the level of the individual bacterial cell along with the actual experimental manipulations. Bacterial cultures each containing a specific BAC clone and the pSC101gbaA plasmid are grown to early log phase at 30 °C (1). The temperature is shifted to 37 °C and expression of the proteins required for recombination is induced with

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-arabinose (2). Cells are washed to remove the medium and the desalted tagging cassettes are electroporated (3,4). After recovery for 1 h (5), cells containing successfully recombined BACs are selected by overnight growth in the presence of kanamycin (6). Incubation at 37 °C also removes the recombination plasmid, which has the temperature-sensitive pSC101 origin of replication. Cm, chloramphenicol; SOC, rich medium for recovery after transformation; Tet, tetracyline; YENB, non-salt medium for electroporation.