Optimized Calcium-Phosphate-Based Co-transfection of Tau and tdTomato into Human iPSC-Derived Neurons for the Study of Intracellular Distribution of Wild-type and Mutant Human Tau.
Methods Mol Biol
; 2754: 551-560, 2024.
Article
en En
| MEDLINE
| ID: mdl-38512689
ABSTRACT
The study of Tau protein in disease-relevant neuronal cells in culture requires efficient delivery systems for transfection of exogenous Tau and also modulators and interactors of Tau. Transfection of cultivated cells using calcium phosphate precipitation is a simple and cost-effective approach, also for difficult-to-transfect and sensitive cells such as primary neurons. Because of its low cell toxicity and ease of use, the Ca2+-phosphate transfection method is one of the most widely used gene transfer procedures in neuroscience. However, Ca2+-phosphate transfection efficacy in neurons is poor, often in the range of 1-5%, limiting its use in functional investigations. Here, we outline our improved Ca2+-phosphate transfection methodology for human iPSC-derived neurons that yields a reasonable efficiency (20-30% for bright volume markers) without apparent effects on cell health. We have used it to introduce wild-type and mutant human Tau with and without co-transfection of a volume marker (used here tdTomato). In sum, our procedure can deliver neuronal genes (e.g., MAPT) using typical eukaryotic expression vectors (e.g., using CMV promoter) and is optimized for transfection of human iPSC-derived neurons.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas tau
/
Células Madre Pluripotentes Inducidas
/
Proteína Fluorescente Roja
Límite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2024
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Estados Unidos