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11Gene Function Analysis Using the ChickenB-Cell Line DT40Randolph B Caldwell, Petra Fiedler, Ulrike Schoetz,and Jean-Marie BuersteddeSummaryQuidquid agis, prudenter agas et respice finem!—Whatever you do, do it wisely and considerthe goal. In consideration of that sage advice, the chicken B-cell line DT40 is an excellent modelcell system to study the function of vertebrate genes. In addition to being highly amenable to genemanipulations, the recent influx of genome and gene/protein resources allows for the straightforwardselection, design, and targeting of candidate genes for knockout analysis. This chapter willgive a step by step standardized protocol to creating a gene knockout mutant in DT40. With carefulconsideration, the methods and protocols described herein can be easily modified to allow forfurther gene manipulations such as creating a knockin or a conditional mutant.Key Words: Conditional mutant; DT40; gene function analysis; gene manipulation; genotype;homologous recombination; knockin; knockout; phenotype; targeting.1. IntroductionAdvantages of DT40 include the high homologous recombination activityfacilitating targeted gene manipulations (1), the availability of tightly regulatedconditional gene expression systems and the ability to study genetic interactionsby the stepwise modification of multiple loci using marker recycling (2).DT40 is particularly suited for genetic analysis if (1) the main phenotype of thegene loss can be studied in cell culture, (2) gene functions are conserved duringevolution, and (3) interactions between multiple genes need to be analyzed andthe genes of interest are essential for murine embryonic development.The recent release of the chicken genome sequence has greatly benefited theDT40 research community (3). For the first time, the genome can be searchedFrom: Methods in Molecular Biology, vol. 408: Gene Function AnalysisEdited by: M. Ochs © Humana Press Inc., Totowa, NJ193
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Gene Function Analysis
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METHODS IN MOLECULAR BIOLOGYGene Fu
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PrefaceThis volume of Methods in Mo
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Prefaceixcolleagues demonstrate how
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xiiContentsPART III EXPERIMENTAL ME
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ICOMPUTATIONAL METHODS I
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4 BidautTable 1Input File Format Us
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6 BidautTable 2Folder Layout to Use
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8 Bidaut• alphaA: this is the num
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10 Bidautcomputing the maximum corr
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Table 3Some Identified Patterns (5,
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16 BidautFig. 4. This is a comparis
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18 BidautReferences1. Hughes, T. R.
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20 Kirov et al.way to associate gen
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22 Kirov et al.based on a study ass
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24 Kirov et al.1. Retrieve the gene
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26Fig. 1. Functional associations f
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28 Kirov et al.Fig. 2. Pathway anal
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30 Kirov et al.3. Gene symbols usag
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32 Kirov et al.9. OBO_Team, Open Bi
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3Estimating Gene Function With Leas
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Estimating Gene Function With LS-NM
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52 Gonye et al.Currently, PAINT can
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Prediction Using PAINT 57represente
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Prediction Using PAINT 59In PAINT,
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Prediction Using PAINT 6114. On the
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Prediction Using PAINT 634.2. Size
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65Fig. 4. Localization of enrichmen
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Prediction Using PAINT 673. Okubo,
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5Prediction of Intrinsic Disorder a
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Prediction of ID and Its Use in Fun
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Table 1Summary of the Web Servers O
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IICOMPUTATIONAL METHODS II
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94 Crabtree et al.genomes, which is
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96 Crabtree et al.Fig. 2. Sybil pro
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98 Crabtree et al.Fig. 3. Computing
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100 Crabtree et al.3.1.5.1. FILTER
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102 Crabtree et al.3. For the sake
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104 Crabtree et al.Fig. 5. Best bid
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106 Crabtree et al.17. Some cluster
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108 Crabtree et al.19. Chado—The
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110 Dateproducts prevents the under
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112 DateDetails of these tasks are
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114 DateThis step creates additiona
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116 Date>hsapiens|gi|20093443 >hsap
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118 DateBLAST score from the match
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Table 1A Sample of Results From Pro
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122 DateFig. 1. A network of functi
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124 Datedescribed by Verjovsky Marc
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126 Dateor contracts put forth by t
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8Bioinformatics Tools for Modeling
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Modeling Transcription Factor Targe
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VISTA Program to search for TFBSs H
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Ac 0 0 0 1 0 1 0 1 0 1 1 0 0 0 0 0
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154 Osborne et al.are included in t
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158 Osborne et al.UMLS source abbre
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160 Osborne et al.Fig. 3. Querying
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162 Osborne et al.3.4.2. Installati
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164 Osborne et al.amount of filteri
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13Construction of Simple and Effici
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DNA Vector-Based shRNA-Expression S
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250 Hust et al.3.4. Titering1. Inoc
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252 Hust et al.10. Shortly before u
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254 Hust et al.activity by preservi
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15A Bacterial/Yeast Merged Two-Hybr
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Screening in Yeast With a Bacterial
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318 Thibodeau-Beganny and Joungbeen
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334 Thibodeau-Beganny and Joung11.
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336 IndexKknockin (gene knockin) 19
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