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)

phox2b:gfp!neuroD:gfp!

Alex Nechiporuk!

BAC transgenics

Kawakami et al.!

Tol2 transposon

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 made by PCR, then simple BP reaction!• LR: overnight in vitro recombination, then transformation!• >99% correct colonies with clear/opaque selection!

entry clones

destination vector

Kristen Kwan, Esther Fujimoto

beta-actin!hsp70!h2afx!CMV/SP6!10x UAS!MCS!

mix and match: Tol2kit entry clones

EGFP!EGFP-CAAX!nls-EGFP!mCherry!mCherry-CAAX!nls-mCherry!H2A-mCherry!Gal4VP16!

polyA!EGFP-pA!mCherry-pA!myc-pA!IRES-EGFP-pA!IRES-EGFP-CAAX-pA!IRES-nls-EGFP-pA!my favorite!

promoter!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

• transient expression from beta-actin promoter shows excellent colocalization between two cistrons!

confocal images of trunk, 24 hpf!

Kristen Kwan

Provost et al., 2007!

Making transgenes visible: viral 2A tagging

nls-mCherry nls-mCherry2A

red green mergeA A’ A’’

B B’ B’’

bactin2: nlsEGFP -IRES-EGFPCAAXpA

bactin2: nlsEGFP -2A-EGFPCAAXpA

EGFPCAAX

IRES vs. 2A

HPAFLYKVGGSGATNFSLLKQAGDVEENPG P9+21 aa C-terminal fusion N-terminal proline

+

C D

nls-mCherry EGFPCAAX

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!

Halloran et al., 2000!

hs for 1 hour at 37°C gives ubiquitous expression!hsp70l promoter

Gal4 enhancer trapping

Scott et al., 2007!

Baier lab enhancer trap screen

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)?!

Issues with Gal4 enhancer traps

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

Pisharath et al., 2007; also Curado et al., 2008!

Metronidazole!conversion!

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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 efficient 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.