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196 Caldwell et al.query because it is likely to be more conserved than the nucleotide sequence indistantly related species. The obtained information might be enough for thedesign of the targeting construct, if the deletion of a conserved region is all that isdesired. As bioinformatics technology grows, the Internet-based tools availablewill reflect the enhanced capabilities as either experimentally known or algorithmicallypredicted sequence structure. The various sites may also show thepredicted 5′ and 3′ noncoding sequences of the transcript, but this information issometimes incomplete or even wrong and it should be considered tentativewithout the support of an experimentally confirmed full-length cDNA sequence.1.1.4. Coding Sequence ConservationThe analysis of the primary amino acid sequence conservation is crucial toanticipate the gene knockout phenotype and to plan its analysis. Most of thecandidate genes are chosen based on the known function of a homolog inother organisms. The higher the primary amino acid sequence conservationof the candidate gene, the more likely it becomes that its function is likewiseconserved. Furthermore, essential structural domains need to be defined forthe construction of targeting vectors if a null mutation is desired, but a completegene deletion is not feasible. Use the amino acid sequence encoded by thechicken candidate gene in a BLAST search of the public protein or EST databases(e.g., http://www.ncbi.nlm.nih.gov/BLAST/) to retrieve the sequencesof the likely homologues from other vertebrate species (e.g., human, mouse,rat, and fish). Use the National Center for Biotechnology Information (NCBI)Conserved Domain Database service to search for conserved domains. ABLAST search of the protein database will automatically identify conserveddomains and will display a link that can be followed for more information onthe identified domains. The usefulness of this and the Gene Ontology (GO,http://www.geneontology.org/) databases should only increase as more islearnt about the relationship that domain structures confer on function.Align and compare the amino acid sequences of orthologs from differentspecies. One way to do so is to copy the sequences of the homologs in FASTAformat to the program “BioEdit” to align and compare the amino acid sequences.BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) is a free software programthat allows the user to import multiple sequences from the various databases,accepts both text and FASTA formats, and has the ability to toggle between nucleicacid and amino acid views. This program is rather easy for the beginner to use andallows for a detailed visual view that is uncomplicated and straightforward.1.2. Design of the Targeting ConstructPhenotypes of DT40 mutants are difficult to predict and in most cases a nullmutation of the candidate gene is aimed for. Deleting the entire gene coding region