Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.
Total time
~3-5 days (including bacterial transformation, selection, and clone analysis)
Model organism
Mouse (transgenic generation)
Steps
1
Design multiplex recombineering oligos
Design oligonucleotide primers with homology arms for simultaneous targeting of multiple genomic sites in the BAC clone. These oligos direct recombination-mediated mutagenesis in E. coli.
▶ 01:04
2
Transform BAC clone into recombineering strain
Introduce the BAC clone into E. coli expressing lambda Red recombination proteins. Prepare competent cells and perform electroporation of the BAC DNA.
▶ 02:00
3
Generate insertion cassettes and subcloning plasmids
Construct donor cassettes containing selection markers and homology sequences. Prepare intermediate subcloning plasmids to facilitate modular assembly of targeting vectors.
▶ 03:56
4
Execute subcloning plus insertion recombineering
Perform sequential recombination events: first insert the cassette into the BAC, then integrate the modified BAC fragment into the final targeting vector plasmid using homologous recombination.
▶ 05:03
5
Analyze recombinant clones by restriction mapping
Isolate plasmid DNA from bacterial colonies and perform diagnostic restriction digestion to confirm correct insertion and proper vector architecture.
▶ 07:07
6
Validate SPI-generated targeting vectors
Present representative restriction patterns and sequencing results confirming successful generation of knockout, knock-in, or tagged allele targeting vectors.
▶ 07:48