In fusion pcr cloning kit

Promoters were PCR-amplified from N₂ genomic DNA and then recombined with specific donor vector fragments using the In-Fusion PCR Cloning Kit (TaKaRa Inc.). The following promoter fragments constructed with GFP or mCherry were used in this study: Psra-6::GFP (ASH neuron) and Pvap-1::mCherry (AMsh glia), which were used to identify ASH neurons and AMsh glia, respectively. Psra-6::GCaMP5.0 and Pvap-1::GCaMP5.0 were used for calcium imaging in ASH neurons and AMsh glia, respectively, as previously described (Ding et al., 2015 (link); Duan et al., 2020 (link)).

DeDFR1 and DeDFR2 were cloned into the expression vector pET28a (+) (Novagen, USA) by using the In-Fusion PCR Cloning Kit (Takara). The PCR primers were designed using In-Fusion Cloning tools (https://www.takarabio.com). Bam HI and Sac I were used as the two restriction sites. The list of primers is provided in S1 Table. In-Fusion cloning was performed according to the manufacturer’s instructions at 50°C for 15 min with a 5-μL reaction system consisting of 1 μL of 5× In-Fusion HD Enzyme Premix, 50 ng of linearized vector and 25 ng of purified PCR fragment. Ligated products were introduced into competent Trans5α cells (Transgen Biology) for sequencing. After confirmation of the sequences, the subcloned vectors were used to transform E. coli strain BL21 (DE3) (Transgen Biology). The empty vector and pET28a-AtDFR were also introduced into E. coli as controls.
The expression of each recombinant DFR protein was induced by 0.2 mM isopropyl-thio-β-D-galactoside (IPTG) in LB culture for 20 h at 20°C. The E. coli cells were harvested by centrifugation and resuspended in extraction buffer (25 mM Tris-HCl, pH 8.0). The cells were lysed by sonication, and the debris was removed by centrifugation at 8,000 rpm for 5 min at 4°C. The protein concentration was estimated by using the Easy Protein Quantitative Kit (Transgen Biology), and the protein quality was examined using 10% SDS-PAGE.

A human hepatocarcinoma cell line (SK-hep1) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). 293T cells and lentiviral vector GV358 were purchased from GeneChem (Shanghai, China). DMEM was purchased from Gibco (CA, USA). The Annexin V-FITC apoptosis detection kit, the NE-PER™ nuclear and cytoplasmic extraction reagents, and thyroid hormone receptor beta-1 antibody were purchased from (Thermo Fisher, MA, USA). Other reagents were obtained as follows: GAPDH antibody (Santa Cruz, CA, USA); the Bcl-2, 4-1BB, Bak, Histone H3, and active Caspase-3 antibodies (Bioss, Beijing, China); the TRIzol total RNA extraction reagent, the In-Fusion™ PCR cloning kit, and quantitative real-time PCR detection kit (Takara, Dalian, China); M-MLV reverse transcriptase (Invitrogen, CA, USA); KOD-Plus-Ver polymerase (TOYOBO, Tokyo, Japan); the Caspase-3 spectrophotometric assay kit (NANJING KEYGEN BIOTECH. CO., LTD, Nanjing, China); and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Promega, Beijing, China). Four-week-old female BALB/c nude mice (15–18 g) were obtained from Shanghai Lingchang BioTech CO., Ltd (Shanghai, China). Protocols involving animals used in this study were approved by the Institutional Animal Care and Use Committee of Weifang Medical University.

Promoters were PCR-amplified from N2 genomic DNA and then recombined with specific donor vector fragments using the In-Fusion PCR Cloning Kit (TaKaRa Inc.). Podr-1:RFP (AWC and AWB), Podr-1:DsRed2b (AWC and AWB), Pstr-2:DsRED (AWC^on), Pstr-1:DsRED (AWB), Psra-6:DsRED (ASH and ASI), Pstr-3:YFP (ASI), Podr-10:mCherry (AWA) and Psrh-220:mCherry (ADL) transgenic strains were used to identify AWC, AWB, ASH, AWA and ADL neurons, respectively. Pstr-1:GCaMP5.0 was constructed for calcium imaging in AWB neurons.

The recombinant NDV strains rTS09-C, rTS-2B, and rTS-GFP (the green fluorescent protein [GFP] gene was inserted into the P-M gene of rTS09-C virus containing three basic amino acids in the FCS) were constructed and rescued previously (17 (link), 48 (link)) and maintained in our laboratory.
For the construction of the full-length cDNA clones of rTS-SS9 and rTS-2B/GFP, strains rTS09-C and rTS-2B were used as the backbone virus, respectively. Briefly, the Tmpress9 gene fragment was amplified from lung tissue of 18-day-old SPF chicken embryos. Tmprss9 and GFP genes were then inserted into the P-M gene of TS09-C and TS-2B, respectively, using an In-Fusion PCR cloning kit (TaKaRa), to generate plasmids pTS-SS9 and pTS-2B/GFP.
To rescue the recombinant NDV, the constructed full-length cDNA clone (pTS-SS9 or pTS-2B/GFP) was cotransfected into MVA-T7-infected BHK-21 cells, with the NP, P, and L supporting plasmids, by using Lipofectamine 3000 (Invitrogen). At 6 h posttransfection, the cells were washed with PBS and cultured in serum-free DMEM, antibiotics, and TPCK-trypsin. The cocultures were incubated for 72 h before inoculation into 10-day-old SPF chicken embryos. At 96 hpi, the allantoic fluids were harvested and the virus was identified by an HA assay and RT-PCR sequencing.