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.

Davidson AE, Balciunas D, Mohn D, Shafer J, Hermanson S, Sivasubbu S, Clif MP, Hackett PB, Ekker SC. Efcient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev Biol. 2003 Nov 15;263(2):191-202.

Emelyanov A, Gao Y, Naqvi NI, Parinov S. Trans-kingdom transposition of the maize dissociation element. Genetics. 2006 Nov;174(3):1095-104.

Emelyanov A, Parinov S. Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish. Dev Biol. 2008 Aug 1;320(1):113-21.

Goll MG, Anderson R, Stainier DYR, Spradling AC, Halpern ME. Transcriptional silencing and reactivation in transgenic zebrafish.

Halloran MC, Sato-Maeda M, Warren JT, Su F, Lele Z, Krone PH, Kuwada JY, Shoji W. Laser-induced gene expression in specific cells of transgenic zebrafish. Development. 2000 127(9):1953-60.

Hatta K, Tsujii H, Omura T. Cell tracking using a photoconvertible fluorescent protein. Nat Protoc. 2006;1(2):960-7.

Huang CJ, Jou TS, Ho YL, Lee WH, Jeng YT, Hsieh FJ, Tsai HJConditional expression of a myocardium-specific transgene in zebrafish transgenic lines. Dev Dyn. 2005 Aug;233(4): 1294-303. Bussmann J, Schulte-Merker S. Rapid BAC selection for tol2-mediated transgenesis in zebrafish. Development. 2011 Oct;138(19):4327-32.

Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell. 2004 Jul;7(1): 133-44.

Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant JM, Yost HJ, Kanki JP, Chien CB.The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.Dev Dyn. 2007 Nov;236(11):3088-99.

Pisharath H, Rhee JM, Swanson MA, Leach SD, Parsons MJ.Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase. Mech Dev. 2007 Mar;124(3):218-29.

Provost E, Rhee J, Leach SD. Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos. Genesis. 2007 Oct;45(10):625-9.

Scott EK, Mason L, Arrenberg AB, Ziv L, Gosse NJ, Xiao T, Chi NC, Asakawa K, Kawakami K, Baier H. Targeting neural circuitry in zebrafish using GAL4 enhancer trapping. Nat Methods. 2007 Apr;4(4): 323-6.

Thermes V, Grabher C, Ristoratore F, Bourrat F, Choulika A, Wittbrodt J, Joly JS. I-SceI meganuclease mediates highly efcient transgenesis in fish. Mech Dev. 2002 Oct;118(1-2):91-8.

Thummel R, Burket CT, Brewer JL, Sarras MP Jr, Li L, Perry M, McDermott JP, Sauer B, Hyde DR, Godwin AR. Cre-mediated site-specific recombination in zebrafish embryos. Dev Dyn. 2005 Aug; 233(4):1366-77.

Villefranc JA, Amigo J, Lawson ND. Gateway compatible vectors for analysis of gene function in the zebrafish. Dev Dyn. 2007 Nov;236(11):3077-87.

Yang Z, Jiang H, Chachainasakul T, Gong S, Yang XW, Heintz N, Lin S. Modified bacterial artificial chromosomes for zebrafish transgenesis. Methods. 2006 Jul;39(3):183-8.