A full-length, human TXNIP plasmid was obtained from Dr Richard T. Lee (Harvard Medical School, Boston, MA, USA) and cloned into a mammalian expression vector (pcDNA3.1; Invitrogen) between the Nhe1 and EcoR1 restriction sites. The plasmid was then propagated in DH5α (Invitrogen) competent cells grown overnight in LB broth (Quality Biological, Gaithersburg, MD, USA) and 50 μg/mL ampicillin (Sigma), and then purified using the HiSpeed plasmid mini-kit (Qiagen). Transformants were analyzed for the presence of the insert with restriction enzyme digestion (New England Biolabs, Ipswich, MA, USA). Exponentially growing Huh7 cells were transfected using Lipofectamine 2000 (Invitrogen), and stably transfected cell lines were selected for in 500 μg/mL Genetecin (Invitrogen), and maintained in 250 μg/mL Genetecin. Expression of TXNIP was confirmed by PCR and immunoblotting. Empty vector was used as a control.
Restriction enzyme digestion
Manufactured by New England Biolabs
Sourced in United States
Restriction enzyme digestion is a core laboratory technique that utilizes enzymes to cut DNA molecules at specific recognition sequences. This process generates DNA fragments that can be further analyzed or modified as part of various molecular biology workflows.
Automatically generated - may contain errors
Lab products found in correlation
Transfast transfection reagent, by Promega (2 mentions)
Genetecin, by Thermo Fisher Scientific (1 mentions)
Hispeed plasmid mini kit, by Qiagen (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Taqman snp genotyping, by Thermo Fisher Scientific (1 mentions)
Nucleospin plasmid easypure, by Macherey-Nagel (1 mentions)
Primestar gxl, by Takara Bio (1 mentions)
Clonexpress multis one step cloning kit, by Vazyme (1 mentions)
5 protocols using restriction enzyme digestion
1
Overexpression of TXNIP in Huh7 Cells
2
Genotyping and Genetic Risk Scoring
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Genotyping was performed as described [10 (link),21 (link)]. Briefly, genomic DNA was extracted from peripheral blood leukocytes by established methods. Genotyping was performed by TaqMan SNP genotyping (Applied Biosystems, Foster City, USA) or by PCR followed by restriction enzyme digestion (New England Biolabs, Ipswich, USA) and subsequent restriction fragment length analysis (RFLP).The resulting genotypes were coded as the number of AMD risk increasing alleles (0, 1 or 2), i.e. alleles which are more frequent in cases than in controls (S1 Table ) [21 (link)]. These variants were used to compute the genetic risk score according to Grassmann et al. 2012 with weights obtained from the parsimonious model based on 10 SNPs (S1 Table ).
3
CRISPR/Cas9 sgRNA Cassette Assembly
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PCR amplification for assembling sgRNA expression cassettes was carried out using PrimeSTAR GXL (Clontech Laboratories, USA). Ligation of sgRNA expression cassettes into the CRISPR/Cas9 vector was performed using the ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd.). The isolation of plasmids was conducted using Nucleospin Plasmid Easy Pure (Macherey Nagel) and analyzed by restriction enzyme digestion (New England Biolabs).
4
Adenoviral Vector Generation Protocol
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The open reading frames of MSTN-SST were ampli ed by PCR using primers listed in Table 1. The PCR amplicons were cloned into a pAd-shuttle-CMV vector. The recombinant adenoviral vectors were generated by homologous recombination of linearized transfer vectors with the pAdEasy-1 in Escherichia coli BJ5183 and con rmed by restriction enzyme digestion (New England Biolabs). The recombinant adenoviruses were generated by transfection of 1 μg plasmids (PacI linearized) using 3 μL of Trans Fast TM Transfection Reagent (Promega, Madison, USA), When 90% of the cells showed cytopathic effect, adenoviruses were released by three cycles of rapid freezing and thawing, and stored at -80 ℃ after addition of 10% glycerol.
5
Recombinant Adenovirus Vector Generation
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The respective open reading frames of fiber-1, fiber-2, and penton base were amplified by polymerase chain reaction (PCR) from viral DNA (FAdV-4 isolate SXD15, refer to KU569296.1) using the primers listed in Table 1. The PCR amplicons of fiber-1, fiber-2, and penton base were not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was this version posted August 17, 2018. ; https://doi.org/10.1101/394072 doi: bioRxiv preprint cloned into a pAd-shuttle-CMV vector. The recombinant adenoviral vectors were generated by homologous recombination of linearized transfer vectors with pAdEasy-1 in E. coli BJ5183 and confirmed by restriction enzyme digestion (New England Biolabs) (49) . The rAds were generated by transfection of 1 µg plasmids (PacI linearized) using 3 µL of Trans Fast TM Transfection Reagent (Promega, Madison, USA). When 90% of the cells showed cytopathic effect, Ads were released by three cycles of rapid freezing and thawing and stored at -80°C after the addition of 10% glycerol.
The copyright holder for this preprint (which was this version posted August 17, 2018. ; https://doi.org/10.1101/394072 doi: bioRxiv preprint cloned into a pAd-shuttle-CMV vector. The recombinant adenoviral vectors were generated by homologous recombination of linearized transfer vectors with pAdEasy-1 in E. coli BJ5183 and confirmed by restriction enzyme digestion (New England Biolabs) (49) . The rAds were generated by transfection of 1 µg plasmids (PacI linearized) using 3 µL of Trans Fast TM Transfection Reagent (Promega, Madison, USA). When 90% of the cells showed cytopathic effect, Ads were released by three cycles of rapid freezing and thawing and stored at -80°C after the addition of 10% glycerol.
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