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Gene Function Analysis Using the Chicken B-Cell Line 197is the safest way to produce such a mutation. Deletions of up to 20 kb have beenintroduced into DT40 by targeted construct integration, but there is the impressionthat the efficiency of targeting is less predictable for very large deletions.Therefore, if the target gene locus is large, then alternative strategies need to beconsidered. A common approach is to introduce an early truncation of the openreading frame in combination with the deletion of a region encoding an indispensablestructural domain. The resulting locus most likely encodes only a shortenedpeptide, and because of the deletion, even aberrant translation or splicing cannotlead to a functional protein. The arms should be amplified by PCR using genomicDT40 as template. This assures that the arm sequences are isogenic to at least oneallele of DT40 that may increase the targeting efficiencies.The plasmid insert of standard DT40 targeting vectors consists of a loxPflanked drug resistance marker cassette flanked 5′ and 3′ by sequences derivedfrom the target locus (see Fig. 1A,B). The 3′ end of the upstream arm and the5′ end of the downstream arm define the boundaries of the target gene deletion.The plasmid is linearized before transfection using a restriction enzyme (RE)(i.e., NotI) whose site is present within the plasmid, but not within the insert.Many of the rules for the design of DT40 targeting vectors are to a certaindegree arbitrary and might be changed if the goal is a single knockout constructfor a particular gene. Nevertheless, following these rules for the design of targetingvectors has the advantage that success rates can be measured for each stepin the vector construction and the subsequent generation of knockout clones.This will give more predictable results and may lead to further optimization ofthe methods presented where needed.1.2.1. The Size and Location of the Target ArmsIf the entire gene-coding region is not larger than 5 kb, the targeting vectoris made by placing the arms upstream and downstream of the coding regionboundaries. This will lead to the deletion of the entire coding region. If it is notpossible to delete the whole gene-coding sequence (e.g., because of its largesize or owing to long intron sequences), the targeting vector is made by placingthe arms upstream and downstream of a coding region that encodes crucialfunctions of the protein. In addition, the downstream primer of the upstreamarm introduces an in-frame stop codon into the gene. The size of the deletion isagain limited to 5 kb. Targeted integration of this vector should lead to a nullmutation as a result of the partial deletion of the coding region and the introductionof the in-frame stop codon (see Fig. 2).The standard sizes of the 5′ and 3′ arms are 3 and 2 kb, respectively.Problems can arise through the presence of restrictions sites that preclude thecloning of the arms, the insertion of the central resistance marker cassette or thelinearization of the targeting vector. If these problems are anticipated, it is first