Retroviral Knockdown of LRRC8A in Breast Cells

LRRC8A silencing with shRNA was performed using retrovirus mediated gene transfer as described previously [4 (link),26 (link)]. To generate retrovirus expressing shRNA against LRRC8A, the target sequences (Table 1) were inserted into the pSUPER.retro.puro retroviral vector (OligoEngine, Seattle, WA, USA). As a control, a non-targeting sequence 5′-ATAGTCACAGACATTAGGT-3′ was introduced. Cloned cell lines stably expressing shLRRC8A showed prominent suppression of LRRC8A mRNA but almost no effects on the expression of other LRRC8 isoforms (LRRC8A-E) in three breast cell lines (Supplemental Figure S1).

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Furuya K., Hirata H., Kobayashi T, & Sokabe M. (2021). Sphingosine-1-Phosphate Induces ATP Release via Volume-Regulated Anion Channels in Breast Cell Lines. Life, 11(8), 851.

Publication 2021

pSUPER.retro.puro retroviral vector

Breast Cell lines Gene transfer Isoforms Mrna Retroviral Shrna Vector

Corresponding Organization : Nagoya University

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Variable analysis

independent variables

LRRC8A silencing with shRNA

dependent variables

Suppression of LRRC8A mRNA
Expression of other LRRC8 isoforms (LRRC8A-E)

control variables

Non-targeting sequence 5′-ATAGTCACAGACATTAGGT-3′

controls

Positive control: Not specified
Negative control: Non-targeting sequence 5′-ATAGTCACAGACATTAGGT-3′

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