Gateway lr clonase reaction

The native CDS of Sr43 plus two additional nucleotides (CC) at the beginning of the CDS (to maintain the open reading frame with His₆ tag) was commercially synthesized (Twist Bioscience) and cloned into the Gateway entry vector pTwist_ENTR. For recombinant protein expression, Sr43 was transferred into the expression vector pDEST-His₆-MBP by Gateway LR clonase reaction (Invitrogen). The resulting clone was verified by Sanger sequencing.
His₆-MBP-Sr43 tagged protein was expressed in the E. coli Rosetta strain by growing the bacterial culture to an optical density OD₆₀₀ of 0.8 at 37 °C and then inducing the protein expression by addition of 0.5 mM isopropyl β-d-thiogalactopyranoside at 18 °C and further incubating the culture for 14–16 h. The recombinant protein was purified under native conditions using Ni-NTA agarose beads (Invitrogen catalog no. R901-15) following the manufacturer’s instructions.

The AM/CBA-EGFP-WPRE-bGH plasmid was kindly provided by Prof. During (Auckland, New Zealand).⁵⁶ (link) The CBA promoter was first described by Niwa et al.⁵⁷ (link) The 1.7-kb chicken β-actin promoter and CMV enhancer is fused to exon 1 (90 bp) of the chicken β-actin gene, a hybrid chicken β-actin and rabbit β-globin intron (917 bp), and rabbit β-globin exon 3 (55 bp), and is also called CB or CAG in the literature. The EGFP reporter gene was removed by BamHI/EcoRV digestion and replaced by a gateway cassette (reading frame A cassette; Invitrogen) from the pBS-Gateway plasmid digested with BglII/EcoRV. Finally, a Gateway LR Clonase reaction (Invitrogen, Thermo Fisher Scientific) was performed with the resulting plasmid (pAAV2ss-CBA-Gateway-WPRE-bGH) and pENTR-AcGFP1nuc (derived from Clontech pAcGFP1-Nuc) to produce pAAV2-CBA-nlsGFP-WPRE-bGH. The pAAV-CMV-EGFP plasmid was obtained by inserting the EGFP cDNA from the pAAV-CMV-MCS plasmid (Agilent, Santa Clara, CA, USA) using standard cloning procedures. The pDP8 (Plasmid Factory, Bielefeld, Germany) or pDP9rs-gck plasmid (kindly provided by Dr. Jurgen Kleinschmidt, Heidelberg, Germany) was used for producing the AAV8 and AAV9 serotypes, respectively.

To generate Rab25b fusion proteins, forward and reverse primers containing attB1 and attB2 BP Clonase recognition arm sequences were used to PCR amplify rab25b. attb-tagged rab25b PCR Products were gel extracted and recombined into a pDONR221 entry vector using BP Clonase (Invitrogen 11789013). Clones were identified via kanamycin selection and validated by sequencing.
pDONR221-rab25b vectors were recombined into a pCS2+ SP6 promoter destination vector by Gateway LR Clonase reaction (Invitrogen 11791020). Destination vectors contained a 5’ SP6 promoter sequence, followed by either eGfp or mCherry with Rab25 integrated in the 5’ to 3’ direction downstream, resulting in a N-terminally tagged Rab25b fusion protein. Sequencing was used to confirm integration and reading frame.

To generate a transgenic rescue construct for Rab25b, forward and reverse primers containing attB1 and attB2 BP Clonase recognition arm sequences were used to PCR amplify rab25b. attb-tagged rab25b PCR Products were gel extracted and recombined into a pDONR221 entry vector using BP Clonase (Invitrogen 11789013). Clones were identified via kanamycin selection and validated by sequencing.
pDONR221-rab25b vectors were recombined into a Tol2 transgenic destination vector by Gateway LR Clonase reaction (Invitrogen 11791020). Destination transgenic vectors contained two Tol2 recognition sequences that flanked divergent ß-actin and myl7 promoter sequences. Rab25b was integrated in the 5’ to 3’ orientation downstream of the ß-actin promoter sequence. Sequencing was used to confirm integration and reading frame. The myl7 promoter sequences contained a downstream RFP expression cassette for screening purposes. To generate Tg(actb1:rab25b,myl7:RFP) fish, Tol2 mRNA (25pg) and the destination vector Tol2-RFP-myl7:ß-actin-rab25b-Tol2 (50pg) were injected into 1-cell stage embryos. Embryos were screened at 48 hpf to confirm Myl7 heart restricted fluorescence and grown to adulthood.

Sequence encoding Cas9, murine Oct4, Sox2, Ddx56, and EGFP were cloned into pENTR. Ddx56 domain truncations were generated by overlap PCR on pENTR-Ddx56. Cas9, Oct4, Sox2, EGFP, Ddx56, and its domain truncations were ligased to pDEST27 or pBabe by gateway® LR clonase reaction (Thermo Fisher) for immunoprecipitation experiments. A17-2loxP mESCs have a doxycycline-inducible promoter and two loxP loci near HPRT gene. Cas9, Ddx56, and its domain truncations were ligated to p2loxP plasmid, then cotransfected with pSalk-Cre plasmid which codes cyclization recombination enzyme (Cre) into A17-2loxP mESCs by Lipofectamine 2000 (Invitrogen). Positive ESC clones were selected by G418 and detected by western blot after adding doxycycline for 48 h. A pair of gRNA oligonucleotides with 5′-CACC and 3′-AAAC overhang was synthesized, annealed, and ligased to vectors (px330, px458, plenti-gRNA1-BSD, and plenti-gRNA2-Hygro). To get inducible knockout Ddx56 cell lines, plenti-gRNA2-BSD and plenti-gRNA2-Hygro were transfected into A17-2loxP-Cas9 cells via retroviral transfection system. The Ddx56 gRNA sequences were as follows: gRNA1, GCCATTCCTCTGGCGCTGG; gRNA2, GTGGTCTGTGAGACAGAAG. Target sites of Ddx56 were PCR amplified using primers in Additional file 1: Table S1. The PCR products were then used in T7 endonuclease I (T7EI) cleavage assay.

The c.571C > T mutation was inserted into a DNAAF1 Gateway entry clone to form a mutant construct by site-directed mutagenesis (SDM) using the Q5 site-directed mutagenesis kit and protocol (New England Biolabs). A SNP of unknown function, c.1634T > G, was removed by SDM. Following this, Gateway LR Clonase reaction (Thermo Fisher Scientific) was performed to subclone mutant and wild-type cDNA into entry vectors pCS2+ and GW331 for SF-TAP. Bacterial transformation and mini/maxi preps (Qiagen) were performed for construct growth and DNA extraction. All cDNA clone sequences were confirmed by Sanger sequencing. Primer sequences available on request.

Plasmids containing the ccdB gene were maintained in One Shot® ccdB Survival™ 2 T1R bacteria (Invitrogen). All enzymes used in this study were purchased from New England BioLabs. All pENTR1 vectors were derived originally from pENTR221-GUS (Invitrogen). gBlocks (Integrated DNA Technologies) encoding the pGG and pACPT cassettes were ordered and constructed to contain attB1/2 sites. Single BP Clonase reactions were performed using manufacturer’s protocols with Clonase II enzyme mixes (Invitrogen). The pT3.5-CAG-Csy4-T2A-hCas9 and pT3.5-CAG-MS2-p65-HSF1-T2A-eGFP vectors were generated using a combination of restriction enzyme cloning and Gateway cloning. Briefly, the nucleotides encoding Pseudomonas aeruginosa Csy4 [5 (link),27 (link)] flanked with attL1 and T2A sites (attL1-Csy4-T2A) with SbfΙ and BspHΙ restriction sites was ordered as a gBlock (Integrated DNA Technologies). The DNA fragment of Csy4-T2A was cloned into pENTR221-hCas9 digested with NcoI. pENTR221-Csy4-T2A-Cas9 was then cloned into pT3.5-CAG-DEST via Gateway LR Clonase reaction, following manufacturer’s instructions (Thermo Fisher Scientific). A gBlock encoding MS2-p65-HSF1-T2A-eGFP flanked by attB1/2 was used for BP Clonase reaction with pDONR221 to generate pENTR221-MS2-p65-HSF1-T2A-eGFP, which was subsequently used for LR Clonase reaction with pT3.5-CAG-DEST to generate pT3.5-CAG-MS2-p65-HSF1-T2A-eGFP.