Guidelines For Successful Gene Targeting
Homologous DNA Fragments
The vector should contain at least 7 kb of effective homologous sequence and the short arm should not be less than 1 kb. For conditional knockout or knock-in projects only the DNA fragments external to the loxP site or to the knock-in element are considered as effective homologous sequence.
The homologous DNA sequence in a targeting vector should match the genomic sequence of ES cells. Isogenic genetic background is recommended. Our facility offers genomic DNA of ES cells for investigators to confirm the genomic map of the targeted gene.
Positive Selection Cassette
The Neomycin resistence gene is the most commonly used positive selection marker but the Hygromycin resistance gene can be an alternative. Recommended promoters include: PGK-1, TK and chicken β-actin.
Negative Selection Cassette
DTA or TK can be used to eliminate some of the ES clones with randomly integrated vector.
DNA Probe for Screening
A probe size of 250 to 300 bp is recommended. A larger size increases the chance of containing repetitive sequence which will result in high background on Southern analysis. The GTF provides ES genomic DNA Southern filter strips on which the quality of the probe can be confirmed to have a high signal to noise ratio.
A physical map of the wild type locus and a map of the expected targeted allele should be worked out by the investigator. All restriction enzymes relevant to the Southern screening purpose have to be confirmed by digestion of ES genomic DNA provided by the GTF and the result has to be shown to the GTF director.