Wild-type GCK-expressing lentiviral vector was generated as previously described [28] (link). Briefly, the mouse GCK encoding sequence (GenBank# BC011139.1) was amplified by PCR and cloned into the BamHI-NotI site of pHRSINCSGW_PGRPuro [29] (link), resulting in pSIN-SFFV-GCK. To generate the V91L GCK-expressing lentiviral vector, the V91L mutation was introduced into the pSIN-SFFV-GCK plasmid by site-directed mutagenesis, resulting in pSIN-SFFV-GCK-V91L. Introduction of the V91L mutation was verified by Sanger sequencing. Lentiviral vector expressing the SV40 small and large T antigens, pLenti CMV/TO SV40 small + Large T, was a generous gift from Dr. Eric Campeau through Addgene (#22298, unpublished; Cambridge, MA, USA)
Plenti cmv to sv40 small large t
Manufactured by Addgene
Sourced in United States
The PLenti CMV/TO SV40 small + Large T is a lentiviral vector that can be used to express SV40 small and large T antigens under the control of a tetracycline-inducible promoter. The vector contains a CMV promoter and a tetracycline operator sequence, allowing for the regulated expression of the SV40 T antigens.
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2 protocols using plenti cmv to sv40 small large t
1
Generation of GCK and GCK-V91L Lentiviral Vectors
2
Establishment of Immortalized Keratinocytes
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Primary keratinocytes obtained from 2-day-old K14Cre.Ptpn2w/w (TC-PTP/WT) or K14Cre.Ptpn2fl/fl (TC-PTP/KO) neonates were cultured in keratinocyte growth medium (PromoCell, #C-20211, C-39011) containing 1% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2 according to the previously described method48 (link). Mouse immortalized keratinocyte cell lines were established by using pLenti CMV/TO SV40 small + large T (Addgene, #22298). Viral packaging using 293T cells and titration of viruses were performed by using ViraPowerTM Lentiviral Packaging Mix (Invitrogen, K497500). The packed virus was concentrated by Lenti-XTM Concentrator (Clontech, 631231). For establishing stable immortalized keratinocyte cell lines, primary keratinocyte cells were infected with lentivirus containing the pLenti SV40 small + large T-antigen-expressing vector. After transduction, cells were cultured with keratinocyte growth medium for over ten passages to select for the clones capable of growing indefinitely.
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