Journal: The Journal of Biological Chemistry

Article Title: Recharacterization of the mammalian cytosolic type 2 ( R )-β-hydroxybutyrate dehydrogenase as 4-oxo- l -proline reductase (EC 1.1.1.104)

doi: 10.1016/j.jbc.2022.101708

Figure Lengend Snippet: Western blot analysis of BDH2 expression in four Bdh2 -deficient HEK293T clonal cell lines ( Bdh2 -KO) and WT HEK293T cells (WT). The KO clonal cell lines (A12, A15, B9, and B13) were generated by the CRISPR–Cas9 gene-inactivation procedure. The Western blot analysis was carried out using 40 μg of the cell lysate protein, a primary rabbit antibody against the human BDH2 (catalog no.: PA5-44760; Invitrogen), and a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody. The secondary antibody was detected by measuring enhanced chemiluminescence. The presence of a nonspecific signal (≈30 kDa) is in agreement with the specification of the primary antibody. BDH2, type 2 ( R )-β-hydroxybutyrate dehydrogenase; HEK293T, human embryonic kidney 293T cell line.

Article Snippet: This vector encodes an enhanced specificity Cas9 enzyme (a gift from Andrea Németh; Addgene; plasmid no.: 101039) ( ).

Techniques: Western Blot, Expressing, Generated, CRISPR

Journal: ACS synthetic biology

Article Title: Temporal Expression of Transcription Factor ID2 Improves Natural Killer Cell Differentiation from Human Pluripotent Stem Cells

doi: 10.1021/acssynbio.2c00017

Figure Lengend Snippet: Targeted gene knock-in in H9 hPSCs for inducible expression of NFIL3, SPI1, and ID2. (A,B) Schematic of all-in-one, Tet-on 3G inducible system construct and targeted knocked-in strategy at the endogenous AAVS1 safe harbor locus via CRISPR/Cas9-mediated homologous recombination shown in panels A and B. (C) PCR genotyping of hPSC clones after puromycin selection is shown, and the expected PCR product for correctly targeted AAVS1 site is 991 bp (red arrow). A homozygosity assay was performed on the knock-in clones, and those without ~204 bp PCR products were homozygous (blue arrow). (D) Flow cytometry analysis of OCT4 and SSEA4 expression in the indicated hPSC lines. (E) RT-PCR analysis of NFIL3, SPI1, and ID2 expression in the indicated hPSC lines with or without doxycycline (dox) treatment.

Article Snippet: Following, 1–2.5 × 10 6 singularized hPSCs were nucleofected with 6 μ g of AAVS1 XLone donor plasmids along with 6 μ g of SpCas9 AAVS1 gRNA T2 (Addgene; #79888) in 100 μ L of human stem cell nucleofection solution (Lonza; #VAPH-5012) using program B-016 in a Nucleofector 2b.

Techniques: Gene Knock-In, Expressing, Construct, CRISPR, Homologous Recombination, Clone Assay, Selection, Knock-In, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction