Short Hairpin RNA-Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based shRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression

Published: April 30, 2023

Abstract

Source: Hsiao, Y. L., et al. Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro. J. Vis. Exp. (2022).

In this video, we demonstrate a method to perform transduction of shRNA lentiviral vectors to obtain stable knockdown cell lines in immortalized hematopoietic stem and progenitor cells (iHSPCs).

Protocol

1. Preparation of immortalized hematopoietic stem and progenitor cell lines (iHSPCs) Maintain iHSPC cell line in complete RPMI 1640 medium containing 100 ng/mL FL and 1 μM β-estradiol. Passage the cells at a ratio of 1:10 every 3 days. NOTE: Make complete RPMI 1640 medium by supplementing with 10% fetal bovine serum (FBS), 5 x10-5 M β-mercaptoethanol, and 10 μg/mL gentamicin. Recombinant murine FL is also commercially available….

Representative Results

Figure 1: The construct of lentiviral vector pLKO.1-Puro.

開示

The authors have nothing to disclose.

Materials

1.5 mL Micro tube  ExtraGene  TUBE-170-C
12-well tissue culture-treated plate  Falcon  353043
Fetal bovine serum (FBS)  Corning  35-010-CV
RPMI 1640 medium  Gibco  11875-085
β-estradiol   Sigma-Aldrich E2758-250MG
β-mercaptoethanol (β-ME)  Sigma-Aldrich  M6250
Flt3 ligand (FL)  Home-made
Polybrene  Sigma-Aldrich  TR-1003-G
Puromycin  Invivogen  ant-pr-1
shId2 (GCTTATGTCGAATGATAGCAA/TRCN0000054390) The RNAi Consortium (TRC)
shLacZ (CGCGATCGTAATCACCCGAGT/TRCN0000072224) The RNAi Consortium (TRC)
shTcf4 (GCTGAGTGATTTACTGGATTT/TRCN0000012094) The RNAi Consortium (TRC)

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記事を引用
Short Hairpin RNA-Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based shRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression. J. Vis. Exp. (Pending Publication), e20969, doi: (2023).

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