Production of CRISPRi-engineered primary human mammary epithelial cells with baboon envelope pseudotyped lentiviral vectors

Sonia Pastor ¹, Julien Wicinski ², Emmanuelle Charafe-Jauffret ², Els Verhoeyen ³, Geoffrey Guittard ⁴, Christophe Ginestier ⁵

STAR PROTOCOL 2023 Jan 19;4(1):102055

doi: 10.1016/j.xpro.2023.102055

 

Primary human mammary epithelial cells (pHMECs) are known to be remarkably difficult to engineer genetically. Here, we present a protocol for efficient transduction of pHMECs using a baboon retroviral envelope glycoprotein for pseudotyping of lentiviral vectors (BaEV-LVs). We describe the preparation of the BaEV-LVs, the isolation of pHMECs from breast samples, and the subsequent transduction of pHMECs. We also detail the use of CRISPRi technology to efficiently silence gene expression in pHMECs, which can then be used for functional assays. For complete details on the use and execution of this protocol, please refer to Richart et al. (2022).¹.