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194 Caldwell et al.for sequences that are conserved during vertebrate evolution between mammalsand chicken. This greatly expedites the identification of worthwhile candidategenes and the subsequent analysis and interpretation of mutant phenotypes.Targeting constructs are now easily derived from the genome sequence to deletegene coding regions, modify regulatory sequences, or add gene coding tags forthe visualization and purification of protein complexes. Furthermore, enhancingthe use of DT40 in gene analysis is (1) the International Chicken PolymorphismMap Consortium’s release of a comprehensive single nucleotide polymorphismanalysis (4), (2) the Second Report on Chicken Genes and Chromosomes 2005(5), and (3) the first serial analysis of gene expression (SAGE) of the chickenB-cell and DT40 genes by Wahl et al. (6). In addition, genes expressed inDT40 are often available as full-length cDNA clones from a large bursalcDNA library, thus enhancing DT40’s usefulness by further facilitating thecomplementation of gene disruption phenotypes and the artificial expressionof proteins (7). Thus, DT40 is well suited to study gene function throughknockout analysis.1.1. The Design1.1.1. Choice of Knockout Candidate GenesCandidate genes for knockouts in DT40 are usually chosen based on structuralhomology to genes with known function in other organisms. A thoroughcheck of what is known about the functions of the homologs and whether thesuspected phenotype can be measured in cell culture is highly recommended atthis stage. Retrieve the nucleotide and amino acid sequences of the nonchickenhomologs either from the public databases (http://www.ncbi.nlm.nih.gov orhttp://www.ebi.ac.uk/embl/) or other sources.1.1.2. Retrieval of Chicken EST or cDNAAlthough an evolutionary conserved cDNA or protein sequence from anonchicken species may yield a positive result in a basic local alignmentsearch tool (BLAST) search against the chicken genome, only knockouts ofgenes expressed in DT40 can be expected to give a phenotype. If there isdoubt of whether the gene is expressed, searches of the Bursal TranscriptDatabase or the bursal and DT40 SAGE tag databases are recommended(http://pheasant.gsf.de/DEPARTMENT/). Careful analysis of ESTs and thecDNA sequence of a knockout candidate gene are also advantageous to definethe exact exon–intron boundaries of the genomic locus. The chicken fulllengthcDNA may also be needed to complement the mutant phenotype.To search for ESTs or cDNAs within The Bursal Transcript Database, enterthe cDNA nucleotide query sequence of the closest homolog from a nonchickenspecies or the chicken cDNA deduced from the chicken genome or other