Gene modification via "plug and socket" gene targeting.

Abnormal hemostasis in a knock‐in mouse carrying a variant of factor IX with impaired binding to collagen type IV

“…The knock‐in targeting construct was designed as a ‘socket and plug construct’ [9]. Our initial FIX knock‐out mouse was generated using a socket construct [10].…”

High-Throughput Site-Directed Mutagenesis in ES Cells

“…Including two copies of the HSV-1 tk gene each under the control of the very strong murine PGK-1 promoter in the selection cassette was supposed to increase the number of Hit clones with at least one functional viral tk gene, thus reducing the number of false positive clones in the Run step. Frequent inactivation of viral genes in ES cells occures due to the susceptibility of viral sequences to gene silencing by methylation (3), by accumulation of point mutations (18), and different codon usage in viral genes also causes proofreading errors resulting in the translation of truncated or inactive viral thymidine kinase (19). However, increased viral thymidine kinase activity from two intact copies of the tk gene persists for several days after the tk genes have been lost.…”

Demystified ... gene knockouts

“…A detailed discussion of other methods of targeting and a detailed description of more advanced methods of conditional targeting have been published elsewhere. [10][11][12][13] The inactivation vector is constructed by the sequential subcloning of fragments, generated by polymerase chain reaction (PCR) of the cloned genomic sequence, into a bacterial plasmid vector, which has the neomycin resistance cassette strategically placed in the plasmid polylinker (multiple cloning site) ( fig 1A and B). An advantage of using PCR to generate the arms of homology is that primer design can be used to induce further mutations in the 1B).…”