Full-length human ASGR1-H1a (GenBank: NM_001197216.2 ), ASGR2-H2b (GenBank: NM_001201352.1 ), and H2c (GenBank: NM_080913.3 ) cDNA sequences were amplified from HepG2 cDNA samples and cloned into a pMSCV retroviral backbone38 (link) by conventional cloning and into a lentiviral backbone containing a puromycin selection marker (kindly provided by Scott Lyons, CSHL) using Gibson Assembly cloning (NEB, Ipswich, MA, USA). Retroviruses and lentiviruses were produced in HEK293T/17 cells by co-transfecting viral constructs with psPAX2 and vesicular stromatitis virus G glycoprotein (VSVG). Viral supernatant was collected 48–72 h post-transfection, filtered, and stored at −80°C. To generate stable cell lines, U87 and HepG2 target cells were infected with lentiviral particles overnight, in the presence of 8 μg/mL polybrene (Sigma, St. Louis, MO, USA), and selected using 1–2 μg/mL puromycin (Sigma, St. Louis, MO, USA) for at least 2 weeks. Cell lines expressing both ASGP-R H1a and H2b isoforms were transduced individually on 2 consecutive days. Retroviral particles were used only for transient experiments.
Puromycin
Manufactured by New England Biolabs
Sourced in United States
Puromycin is a laboratory reagent used in molecular biology and cell culture applications. It functions as a selective agent, enabling the identification and enrichment of cells that have successfully integrated a desired genetic construct. Puromycin inhibits protein synthesis, leading to the death of cells that do not express the resistance gene included in the genetic construct.
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Lab products found in correlation
2 protocols using puromycin
1
Generating Stable Cell Lines with ASGP-R Isoforms
2
CRISPR Plasmid Design and Construction
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pCMV-hA3A-BE3-Y130F used in this study was reported previously [20 (link)] and purchased from Addgene (www.addgene.org/113428 , accessed date 20 August 2018). pGL3-U6-sgRNA-PGK-puromycin used in this study [22 (link)] was reported previously and purchased from Addgene (www.addgene.org/51133 , accessed date 2 March 2014). SgRNA oligonucleotides targeting the TP53, PTEN, and APC genes (Figure 1 a–c) were designed using online software (http://crispor.tefor.net/ , accessed date 20 August 2018), and annealed and cloned into the pGL3-U6-sgRNA-PGK-puromycin plasmid after BsaI (NEB, R0535S) digestion. All plasmid DNA was extracted using EndoFree® Plasmid Maxi Kit (Qiagen, Hilden, Germany).
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