Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign

• Gene Targeting Constructs
• Homology Arms

Gene targeting contructs are designed to undergo homologous recombination into a specific locus chosen by the investigator, usually with the aim of disrupting the gene to prevent transcription of a functional mRNA (a knock-out), or mutating the gene (a knock-in).

The number of ways in which gene targeting constructs can be designed to produce knock-out or knock-in is almost limitless. Thus, we can only offer some general guidelines here, and urge people making their first construct to read the literature and consult with the TMF or another more experienced investigator before doing any cloning.

The simplest targeting construct consists of 2 long segments of genomic DNA (gDNA), called homology arms, flanking a selection cassette. The most commonly used selection cassette consists of the cDNA and control elements for the neomycin (G418) resistance gene (others include resistance genes for puromycin, hygromycin, and 6-thioguanine).

When introduced into the cell line, the gDNA homology arms will undergo recombination with their matching sequences on one chromosome, carrying the selection cassette with them. The gDNA between the regions of homology on the chromosome is thereby replaced by the selection cassette and any other sequences flanked by the homology arms of the targeting construct. Where a complete knockout is desired, the intervening sequence is usually positioned to replace the TATA box, the start codon, and one or more of the initial exons.

The targeting construct will also integrate into random loci. Any integration event, random or specific, can confer drug resistance to the cell. After growing the transfected cells under selection, the challenge is to screen enough clones to find the rare homologous recombination events in a background of frequent random integrants.

We recommend the use of both positive and negative selection cassettes in all targeting constructs. The most commonly used negative selection cassette contains the gene for thymidine kinase, or tk. The tk gene product allows growing cells to incorporate a toxic nucleotide analog into their DNA, thus selecting against those cells. The tk cassette is cloned into the targeting construct outside of the homology arms, so that it will not be incorporated during homologous recombination. It will be incorporated during random integration and help to select against those clones. Another negatively selectable marker is the gene for diphtheria toxin A. The A subunit inhibits protein synthesis but cannot be taken up by other cells. Its advantage over tk is that it works without having to add a second drug to the culture medium.

The degree to which the homology arms match the same sequences in the locus of interest will help determine the frequency of homologous recombination. The 3 most important characteristics of homology arms are:

1. Length – we recommend an overall length of about 7 kilobases, with one arm being 5-6 kb and the other being 1-2 kb. Longer is better, but one is usually limited by the capacity of the cloning vector and the need to maintain a unique restriction enzyme site that can be used to linearize the construct prior to transfection into the cells.


2. Sequence homology – whenever possible, clone the homology arms from the genome of the cells that will be targeted. Long-range PCR with a high-fidelity polymerase is an effective method for subcloning the homology arms.


3. Limited repetitive sequences – we recommend using the on-line program, RepeatMasker, to search for repetitive sequences in the homology arms. Large regions of repetitive DNA should be avoided, because these will result in a lower frequency of homologous recombination.