TgBAC(wt1b:RTTA-2A-CreERT2)

ID

ZDB-TGCONSTRCT-200623-1

Name

TgBAC(wt1b:RTTA-2A-CreERT2)

Previous Names

• TgBAC(wt1b:rtTA-p2A-creERT2) (1)

Type

engineered_plasmid

Regulatory Regions

• wt1b (1)

Coding Sequences

• 2A (1)
• Cre (1)
• ERT2 (1)
• RTTA (1)

Contains Sequences

• TOL2

Note

The construct to generate the transgenic line TgBAC(wt1b:rtTA-p2A-creERT2)cn19 (referred to in the text as wt1b:creERT) was generated by BAC recombineering using EL250 bacteria166. Fragments were amplified by PCR, adding 50 nucleotide homology arms. First, the iTol2Amp-γ-crystallin:RFP cassette162 was amplified using primers 1. pTarBAC_HA1_iTol2_F 5′-gcgtaagcggggcacatttcattacctctttctccgcacccgacatagatCCCTGCTCGAGCCGGGCCCAAGTG-3′ and pTarBAC_HA2_iTol2CrystRFP_R 5′-gcggggcatgactattggcgcgccggatcgatccttaattaagtctactaTCGAGGTCGACGGTATCGATTAAA-3′ and recombined into the backbone of the BAC clone CH73-186G17 to replace the BAC-derived loxP site. Then, the rtTA-p2A-iCreERT2 cassette was amplified and recombined replacing the ATG of the wt1b coding sequence, with primers wt1b_HA1_rtTA_F 5′-gacattttgaactcagatattctagtgttttgcaacccagaaaatccgtcACCATGGTCGACGCCACAACCAT-3′ and wt1b_HA2_FRT_R 5′-gcgctcaggtctctgacatccgatcccatcgggccgcacggctctgtcagGGAGGCTACCATGGAGAAG-3′. Finally, the Kanamycin resistance cassette was removed by inducing expression of Flipase recombinase in the EL250 bacteria. The final BAC was purified with the HiPure Midiprep kit (Invitrogen) and injected along with synthetic Tol2 mRNA into wildtype strain zebrafish embryos. Sequence information and primer details are freely available upon request