2.5 kb of human VASA upstream of the first codon was cloned into pENTR 5′-TOPO. eGFP was fused 1kb downstream of the last codon of human VASA, and cloned into pENTR/D-TOPO. Cloned plasmids were recombined27 (link) to create pLVGV. Lentiviral supernatant was produced, hESCs were transduced overnight on matrigel in conditioned medium and subsequently selected with geneticin (200ng/ml) for 7 days. Selected hESCs were differentiated for times indicated and harvested by brief treatment with Collagenase IV and then TrypLEExpress (Invitrogen). The cell suspension was prepared in differentiation medium for FACS with a MoFlow or BD cell sorter.
Protocol detail
VASA Promoter-driven eGFP Tracking
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Brief treatment
Cell
Codon
Collagenase
Conditioned medium
Geneticin
Hescs
Human
Matrigel
Plasmids
Topo
Corresponding Organization :
Other organizations : Stanford University
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Variable analysis
independent variables
- Cloning of 2.5 kb of human VASA upstream of the first codon into pENTR 5′-TOPO
- Fusion of eGFP 1kb downstream of the last codon of human VASA, and cloning into pENTR/D-TOPO
- Recombination of cloned plasmids to create pLVGV
- Lentiviral supernatant production
- Transduction of hESCs with lentiviral supernatant overnight on matrigel in conditioned medium
- Selection of transduced hESCs with geneticin (200ng/ml) for 7 days
- Differentiation of selected hESCs for times indicated
- Harvesting of differentiated cells by brief treatment with Collagenase IV and TrypLEExpress
- Preparation of cell suspension in differentiation medium for FACS
- Matrigel used for hESC culture
- Conditioned medium used for hESC culture
- No positive or negative controls were explicitly mentioned in the provided information.
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