Pureyield plasmid miniprep system

Primers used for gene cloning were purchased from Hokkaido System Science. Coding sequences of GV genes were PCR-amplified with 5′ primers encoding a NheI site and 3′ primers encoding an EcoRI site without termination codons using KOD-plus-Neo (TOYOBO). T2A-fluorescent protein (mKate2, mKO2, and EGFP) fusion genes were also PCR amplified with 5′ primers encoding an EcoRI site and 3′ primers encoding a NotI site. A Tol2 cloning vector (donated by Dr. Akira Takai of RIKEN and described in detail previously [15 (link)–17 (link)]) was also digested in the same way. The PCR products and restriction enzyme digestions were purified by agarose gel electrophoresis followed by processing with the Wizard SV Gel and PCR cleanup system (Promega). Restriction enzymes were purchased from Fermentas. The digested PCR products of GV genes and T2A-fluorescent protein fusion genes were ligated to the vectors using Ligation high Ver.2 ligase (TOYOBO) following the recommended protocol of the manufacturer. Plasmids were prepared from the bacterial liquid culture by using the Pure Yield Plasmid Miniprep System (Promega) and PureLink HiPure Plasmid Midiprep Kit (Invitrogen). The DNA sequences were read by dye terminator cycle sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

Penicillin-Streptomycin (P0781), PMA (P8139), βME (M3148), Hank’s balanced salt solution (H9269), DPBS (D8537) and polybrene (107689) were purchased from Sigma Aldrich. DMEM (BE12-604F, Lonza), Trypsin/EDTA (BE17-161E, Biowhittaker), FBS (10270–106, GIBCO) and LPS-EK Ultrapure from the E. coli K12 strain (tlrl-peklps) from Invivogen. Opti-MEM I Reduced Serum Medium (11058021) from ThermoFisher Scientific and Puromycin (540222) from VWR. BsmB1 restriction enzyme (R0580), T4 DNA ligase (M0202) and T4 DNA ligase reaction buffer (B0202) were from New England Biolabs. PureYield Plasmid Miniprep System (A1222) was from Promega.

Plasmid DNA was extracted from E. coli using the PureYield Plasmid Miniprep System (Promega, Madison, WI). E. coli cultures were grown overnight in LB medium supplemented with the appropriate resistance markers. DNA was quantified by the Qubit dsDNA HS Assay (Invitrogen, Carlsbad, CA). Each plasmid was diluted to 0.1 ng/μl for use in library construction. Plasmids are listed in Table 1. All plasmids used are available through the public instance of the ABF registry [18 (link)]. See the availability of data and materials section for additional information.

Plasmids were constructed using standard molecular techniques. Plasmid DNA was isolated using the PureYield Plasmid MiniPrep System (Promega, Madison, WI). Q5 DNA polymerase (New England Biolabs, Ipswich, MA) was used to amplify DNA with primers synthesized by Integrated DNA Technologies, Coralville, IA or Eton Bioscience, Inc., Research Triangle Park, NC. PCR products were purified using the PCR purification kit (Qiagen, Venlo, Limburg, The Netherlands). Restriction endonucleases were purchased from New England Biolabs, Ipswich, MA, and ligase was purchased from ThermoScientific, Waltham, MA.

For synthesis of Cre and Dre mRNAs, the expression vectors pT3TS-Cre (Clark et al., 2011 (link)) and pT3TS-Dre were linearized with SalI (New England Biolabs, R0138S) and BamHI (New England Biolabs, R0136S), respectively. 1 μg linearized vector was purified with the PureYield Plasmid Miniprep System (Promega, A1223) and used as template for in vitro synthesis of capped mRNA with the Ambion mMessage Machine T3 Transcription Kit (Thermo Fisher, AM1348). In vitro synthesized mRNA was purified with the RNA Clean and Concentrator Kit RCC (Zymo, R1013). A total of 12.5 pg Cre or 15 pg Dre mRNA was injected into one-cell stage embryos to promote recombination-mediated inversion of the UFlip cassette at lox or rox sites. UFlip cassette inversion in Cre or Dre injected 3 dpf larvae was confirmed by digestion of individual larvae in 50 mM NaOH at 95 °C for 30 min and neutralization by addition of 1/10^th volume 1 M Tris-HCl pH 8.0. Genomic DNA/UFlip cassette 5’ and 3’ junctions were amplified by PCR with gene specific and UFlip primers listed in Table 4, followed by direct Sanger sequencing.