Transgenics (I): how to make them Stable transgenesis: !! !• Tol2! !• BAC transgenesis ! ! Making transgenesis vectors:! !• Gateway-based methods !! ! !! Other methods:! !• Ac/Ds (Maize), Sleeping Beauty, !Emelyanov et al., 2006 " Tol1, other transposons? ! !Davidson et al., 2003! !• I-SceI ! !Thermes et al., 2002! ! Transgenesis methods Linear or circularized DNA !most mosaic; inserts as rearranged concatamers;" transgenesis rate ~1%! ! DNA linearized with I-SceI !less mosaic; sometimes inserts as single-copy; transgenesis rate ~10%! ! BACs (linear) !mosaic; smaller concatamers; 1% (variable); iTol2, 20%+! ! Transposons (Tol2 etc.) !least mosaic; usually inserts as clean single copies; transgenesis rates of 30-50%! ! Plasmid transgenesis isl2b:gfp! Andrew Pittman! Transgenesis problems Driving expression in desired cells/tissues! !• find appropriate enhancer/promoter! !• avoid ectopic expression! !• BACs can be a solution! ! Silencing/variegation! !• depends on construct sequence! !• made worse by concatamerization! !• depends on insertion position! BAC recombination (recombineering) BAC transgenics phox2b:gfp! neuroD:gfp! Alex Nechiporuk! Tol2 transposon Kawakami et al.! iTol2: inverse tol2 for BACS Bussmann and Sculte- Merker 2011 the Tol2kit: a system for rapid construction of transgenesis vectors • transgenesis efficiency => Tol2! • tedious conventional subcloning => 3-part multisite Gateway, multiple pretested standard elements! • difficulty detecting nonfluorescent transgenes (hs or UAS; Gal4, etc.) => IRES markers, cmlc2:egfp transgenesis marker in backbone! more info: http://chien.neuro.utah.edu/tol2kitwiki/! The 3-way LR reaction is very efficient entry clones destination vector • entry clones made by PCR, then simple BP reaction! • LR: overnight in vitro recombination, then transformation! • >99% correct colonies with clear/opaque selection! Kristen Kwan, Esther Fujimoto mix and match: Tol2kit entry clones beta-actin! EGFP! polyA! hsp70! EGFP-CAAX! EGFP-pA! h2afx! nls-EGFP! mCherry-pA! CMV/SP6! mCherry! myc-pA! 10x UAS! mCherry-CAAX! IRES-EGFP-pA! MCS! nls-mCherry! IRES-EGFP-CAAX-pA! my favorite! H2A-mCherry! IRES-nls-EGFP-pA! promoter! Gal4VP16! my favorite! gene! Kwan, Fujimoto, et al. Transgenics (II): how to identify/propagate them Tracking transgenes:! •# linked transgenesis marker! •# IRES! •# PTV viral 2A peptide! Making transgenes visible: cmlc2:gfp Two transgenes act independently:! • cmlc2:gfp marker drives only in heart, not after heat-shock! • hsp70l:mCherry-CAAX drives only after HS, not in heart! Kristen Kwan Making transgenes visible: EMCV IRES confocal images of trunk, 24 hpf! • transient expression from beta-actin promoter shows excellent colocalization between two cistrons! Kristen Kwan Making transgenes visible: viral 2A tagging Provost et al., 2007! nls-mCherry 2A EGFPCAAX nls-mCherry HPAFLYKVGGSGATNFSLLKQAGDVEENPG + P EGFPCAAX 9+21 aa C-terminal fusion N-terminal proline red green merge A A’ A’’ IRES vs. 2A bactin2: nlsEGFP - bactin2: nlsEGFP - IRES-EGFPCAAXpA 2A-EGFPCAAXpA C D B B’ B’’ Kristen Kwan Transgenics (III): conditional or tissue-specific expression heatshock! Gal4/UAS! ! Others: ! !! !• Cre/Lox !Thummel et al., 2005 ! !! !• Tet on/off !Huang et al., 2005! !• ecdysone receptor !Esengil et al., 2007! !• mifepristone/PR !Emelyanov and Parinov, 2008! hsp70l promoter hs for 1 hour at 37°C gives ubiquitous expression! Halloran et al., 2000! Gal4 enhancer trapping Baier lab enhancer trap screen Scott et al., 2007! Issues with Gal4 enhancer traps Toxicity of Gal4VP16 overexpression! !• modified Gal4s (Gal4FF)! Basal/background expression! !• different minimal promoters! Variegation! !• UAS prone to methylation (Goll et al., 2009)! !• improved but not eliminated by Tol2! Position effects! !• development of site-specific integration (phi31c)?! Transgenics (IV): what to express Genes of interest! ! Activatable fluorescent proteins! ! Calcium reporters (or FRET-based reporters)! ! Activity modifiers! • ion channels! • tetanus toxin! ! Photoactivatable channels (channelrhodopsin)! ! Cell killing (nitroreductase)! UAS:Kaede Parsons et al., 2006! Hatta et al., 2006! Nitroreductase ablation Metronidazole! conversion! Pisharath et al., 2007; also Curado et al., 2008! References! Asakawa K, Kawakami K. Targeted gene expression by the Gal4-UAS system in zebrafish. Dev Growth Difer. 2008 Aug;50(6):391-9. Curado S, Stainier DY, Anderson RM. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat Protoc. 2008;3(6):948-54. 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