Lentiviral Infection of Mammosphere Cultures

To infect mammosphere cultures with lentivirus, primary mammospheres were disassociated as for secondary mammosphere assays. Before plating, single cell suspensions in DMEM/F12 media were mixed with lentiviral particles in PBS at a multiplicity of infection of greater than 5∶1, plus a 1∶200 dilution of Transdux reagent (System Biosciences) for 30 minutes at 37°C. After infection, the cell suspension was plated as for secondary sphere assays. The 7TGC lentiviral reporter vector was obtained from Addgene (Plasmid#24304) [49] (link). Lentiviral vectors for knockdown of Ror2 mRNA were obtained from Dr. T. Stappenbeck [50] (link). For clarity we renamed the knockdown vector shRor2#7 [50] (link) as shRor2 and the control vector SCH002-EGFP as shControl.

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Many A.M, & Brown A.M. (2014). Both Canonical and Non-Canonical Wnt Signaling Independently Promote Stem Cell Growth in Mammospheres. PLoS ONE, 9(7), e101800.

Publication 2014

Transdux reagent

Assays Cell Dilution Infection Lentivirus Mrna Plasmid Ror2 Vector

Corresponding Organization :

Other organizations : Cornell University

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Variable analysis

independent variables

Lentiviral particles (multiplicity of infection greater than 5:1)
Transdux reagent (1:200 dilution)

dependent variables

Mammosphere formation

control variables

Cell suspension plating (as for secondary sphere assays)
Infection time (30 minutes at 37°C)

controls

Positive control: 7TGC lentiviral reporter vector (Addgene Plasmid #24304)
Negative control: shControl (SCH002-EGFP)

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