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MB Sample ID: SA241926

Local Sample ID:C220181610_PKR_Null_5
Subject ID:SU002501
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:Spleen-derived macrophage from WT or Eif2ak2-/-
Age Or Age Range:NA
Weight Or Weight Range:NA
Height Or Height Range:NA
Gender:Not applicable

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Collection:

Collection ID:CO002494
Collection Summary:Exogenous genes were stably expressed and endogenous gene expression suppressed in murine macrophages by transduction with lentiviral constructs. Gene open reading frames were cloned as NheI-BamHI (New England Biolabs) fragments in the plentiCRISPR v2-Blast (Mohan Babu, Addgene plasmid # 83480; http://n2t.net/addgene:83480; RRID: Addgene_83480). Knockdown of endogenous transcripts was achieved with short hairpin RNA cloned into pLKO.1-puro. Lentiviral particles were packaged using a Lenti-X mix (Takara) by transfection of the plasmid components into HEK293FT cells with Lipofectamine 2000 (Invitrogen). The culture supernatant was collected after 48 hours, centrifuged at 10 000 g, then pipetted onto adherent macrophages. Transduced cells were isolated over 2-3 month by increasing selection (from 1 to 20 ug/mL) with puromycin (Thermo Fisher Scientific) and/or blasticidin (InvivoGen), for pLKO-1-puro-shRNA and plentiCRISPR v2-blast, respectively. Recombinant cells were maintained with antibiotics in Dulbecco’s modified Eagle’s Medium (DMEM, GibCo) supplemented with 10% foetal bovine serum (Bovogen) in a humidified 5% CO 2 incubator at 37 °C. Growth on different carbon sources was tested by plating 6x10 3 cells per well of a 24 well plate with glucose free DMEM (ThermoFisher Scientific) supplemented with 10% FBS (not dialysed), 20 ug/mL puromycin and 20 uM of each sugar. After 72 hours the cells were lifted with trypsin and counted by automation (Countess, Invitrogen).
Sample Type:Macrophages
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