Genetic Modification of Primary Human Keratinocytes: A Method for Genetically Manipulating Keratinocytes Using Recombinant Retroviruses
Published: April 30, 2023
Abstract
Source: Li, J. et al. Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis. J. Vis. Exp. (2015)
This video describes a detailed protocol for producing genetically modified primary human keratinocytes recombinant retrovirus. This method can be used to manipulate gene expression in keratinocytes.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Genetically Modifying Primary Human Keratinocytes NOTE: Use amphotropic phoenix cells grown in complete DMEM media [DMEM + 10% fetal bovine serum (FBS) + pen/strep] to produce viruses to infect primary human kera…
Disclosures
The authors have nothing to disclose.
Materials
| Amphotropic phoenix cell lines | ATCC | CRL-3213 | |
| FUGENE 6 transfection reagent | Promega | E2691 | |
| Keratinocyte Media (KCSFM) | Life Technologies | 17005042 | |
| DMEM | GIBCO | 11995 | |
| human neonatal keratinocytes | ATCC | PCS-200-010 | |
| human neonatal keratinocytes | Cell Applications | 102K-05n | |
| MSCV retroviral vector | Clontech | 634401 | |
| LZRS retroviral vector | Addgene | ||
| pSuper.Retro.Puro Retroviral vector |
Oligoengine | VEC-PRT-0002 | |
| hexadimethrine bromide | Sigma | H9268-5G | |
| FBS | GIBCO | 10437-028 | |
| PEN/STREP | GIBCO | 15140-122 |
Cite This Article
Genetic Modification of Primary Human Keratinocytes: A Method for Genetically Manipulating Keratinocytes Using Recombinant Retroviruses. J. Vis. Exp. (Pending Publication), e20360, doi: (2023).