Plenti pgk neo dest

The lentiviral plasmid vector expressing TERT and puromycin acetyltransferase (pac) (pSD-83) was described by Duss et al [12 (link)]. The vector expressing BMI1 and CFP (pER7)
was constructed from pSD-84 [12 (link)] by
replacing the pac gene with CFP by standard cloning. The vector expressing
MYC and hygromycin phosphohydrolase
(hph) (pSV32) was constructed by shuffling
the MYC open reading frame from pSD-94
[12 (link)] into pLenti PGK-hygro DEST
(Addgene 19066) by Gateway cloning. The vector expressing CCND1 and neomycin phosphotransferase (npt) (pSV31) was constructed by Gateway cloning of the CCND1 open reading frame from pENTR-CCND1 (PlasmID
HsCD00001252) into pLenti PGK-Neo DEST (Addgene 19067). The pLVTH-sip53 vector
expressing GFP and a short hairpin RNA
targeting TP53 was obtained from Addgene
(12239). The vector expressing PIK3CA-H1047Rand hph (pER157) was constructed by cloning the
PIK3CA-H1047R open reading frame from
pBABE-PIK3CA (PlasmID clone 25920) into pENTR1A to give pER152, then transferred
by Gateway cloning into pLenti CMV/TO-hygro DEST (Addgene 17484). The vector
expressing tdTomato (pER5) was kindly provided by Francois Moreau-Gaudry.

Human Plexin-B2 cDNA (clone HsCD00399262, DNA Resource Core at Harvard Medical School) was modified by site-directed mutagenesis (Phusion SDM kit, Invitrogen) to generate CRISPR-resistant synonymous cDNA variant. Next, mutations in specific domains of Plexin-B2 were generated by SDM as follows: mGAP (mutation of GAP domain: R1391A, R1392G), mRBD (mutation of RBD domain: LSK 1558–1560 −> GGA), ∆VTDL (deletion of C-terminal amino acids of PDZ-binding motif), and ∆Ecto (a truncated variant that lacks extracellular domain by deletion of amino acids 30–1189). These constructs were then cloned into lentiviral vector backbone pLENTI PGK Neo DEST (Addgene 19067; deposited by Eric Campeau & Paul Kaufman) by Gateway. GSCs were infected with the Plexin-B2 cDNA lentiviruses as described above, and transduced cells were selected with 150 µg/ml G418 (Gibco).