A codon-optimized gene encoding the c-Met-specific single-chain variable fragment (scFv) of human c-Met Fab was amplified[15 (link)], and the amino sequences for this scFv are provided in Supplementary Table 1 (available online). The optimized sequence contained a heavy chain variable region-(GGGS)3-light chain variable region sequence. The sequence was subcloned in-frame into lentiviral vectors containing expression cassettes encoding a CD8α hinge domain, CD8 transmembrane (TM) domain and CD28-CD3ζ, CD137-CD3ζ, or CD28-CD137-CD3ζ signaling domains. The sequence of each cloned CAR was verified by DNA sequencing (Genscript, China). The c-Met CAR fragments (c-Met-28-3ζ, c-Met-137-3ζ, and c-Met-28-137-3ζ) were generated by PCR using the following primers: forward, 5′-GGACCATCCTCTAGGGATCCATGGCCTTACCAG-3′; and reverse, 5′-AATCCGGATCGATCTCGAGGTGCATGCTAACGC-3′. They were cloned into the BamH 1 and Xho 1 sites of the lentiviral vector pCDH-CMV-MCS-EF1α-CopGFP[15 (link)]. The new vector was verified by DNA sequencing (Genscript). In addition to activated T cells, a CD19-specific CAR containing CD137 costimulatory endodomains and CD3ζ was used as a negative control, and the detailed structure has been described previously[16 (link)].
Dna sequencing
Manufactured by GenScript
Sourced in China
DNA sequencing is a laboratory technique used to determine the precise order of the four chemical building blocks (adenine, guanine, cytosine, and thymine) that make up a DNA molecule. This process provides essential information about the genetic makeup of an organism or a specific DNA sample.
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Lab products found in correlation
Phoenix ampho 293t packaging cell line, by GenScript (1 mentions)
Plcnx2, by GenScript (1 mentions)
Gene pulser apparatus, by Bio-Rad (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Easypure plasmid miniprep kit, by Transgene (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
5 protocols using dna sequencing
1
Construction of c-Met-Specific CAR T Cells
2
Lentiviral and AAV9-Mediated Gene Transduction
3
Construction of FADD and N-FADD Plasmids
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N-FADD (m-FADD, 1–181 aa), which is truncated of the C-terminal tail of mouse FADD, was amplified by primers N-FADD-F and N-FADD-R with pcDNA-FADD as template. Primers sequences as follows:
N-FADD-F: 5′-CGGGATCC ATGGACCCATTCCTGGTGC-3′
N-FADD-R: 5′-CCCAAGCTT TCACTGGGCTTCTTCCACCAG-3′
The sequence was cloned into eukaryotic expression vector pcDNA3.1 (−) after digested with BamHI and HindIII. The positive clone contains sequence of N-FADD named pcDNA-N-FADD. For hypoxia-inducible expression in VNP, the sequences of FADD and N-FADD were inserted into the prokaryote expression vector pQE30-NirB with BamHI, HindIII digestion and the resultant vectors were named pQE-NirB-FADD and pQE-NirB-N-FADD, respectively. All of the coding sequences of positive clones were confirmed to be correct by DNA sequencing (GenScript Corporation, Nanjing, Jiangsu, China). pQE-NirB, pQE-NirB-FADD and pQE-NirB-N-FADD plasmids were transformed into VNP using a Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA) with conditions as follows: 2.5 kV, 25 μF and 400 Ω, respectively and plated on LB agar containing 100 μg/ml ampicillin. Positive VNPs were each named VNP-pN, VNP-pN-FADD and VNP-pN-N-FADD.
N-FADD-F: 5′-CG
N-FADD-R: 5′-CCC
The sequence was cloned into eukaryotic expression vector pcDNA3.1 (−) after digested with BamHI and HindIII. The positive clone contains sequence of N-FADD named pcDNA-N-FADD. For hypoxia-inducible expression in VNP, the sequences of FADD and N-FADD were inserted into the prokaryote expression vector pQE30-NirB with BamHI, HindIII digestion and the resultant vectors were named pQE-NirB-FADD and pQE-NirB-N-FADD, respectively. All of the coding sequences of positive clones were confirmed to be correct by DNA sequencing (GenScript Corporation, Nanjing, Jiangsu, China). pQE-NirB, pQE-NirB-FADD and pQE-NirB-N-FADD plasmids were transformed into VNP using a Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA) with conditions as follows: 2.5 kV, 25 μF and 400 Ω, respectively and plated on LB agar containing 100 μg/ml ampicillin. Positive VNPs were each named VNP-pN, VNP-pN-FADD and VNP-pN-N-FADD.
4
Avian HMBS-based qPCR Specificity Validation
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The specificity of the avian HMBS-based qPCR was verified using DNA from whole blood of seven chickens indigenous to China (Qingke chicken, Lanya Baitiao chicken, Luhua chicken, Rugao Huang chicken, Huxu chicken, Qingguan chicken, Baier chicken; kindly provide by the Poultry Institute of China), four rare bird species (wild goose, quail, swan, crane; kindly provided by the Yangzhou Zoo), a duck, goose, and pigeon, as well as DNAs from seven mammalian species (human, cat, dog, cattle, goat, sheep, and pig). PCR products were further verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA), DNA sequencing (GenScript, Nanjing, Jiangsu, China) and BLASTn.
5
Constructing CaHEV ORF3 Standard Plasmid
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A fragment containing the complete sequence of the CaHEV ORF3 gene was amplified using the F2 and R2 primers to prepare the standard control plasmid. The RT-PCR products were then cloned into the pMD 18-T vector according to the manufacturer’s instructions (TaKaRa Biotech Corporation, Dalian, China). The standard plasmid DNA was extracted from transformed Escherichia coli DH5α competent cells using the EasyPure Plasmid MiniPrep Kit (Transgen Biotech Corporation, Beijing, China) and confirmed with DNA sequencing (Genscript Biotech Corporation, Nanjing, China). DNA concentration was determined by spectrophotometry (Epoch) at 260 nm, and the average concentration was determined using five independent measurements. The copy number of the recombinant plasmid was calculated using the following formula: (DNA concentration in ng/μl×10−9×6.0233×1023 copies/mol)/[DNAsize (bp)×660] [21 (link)].
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