Enhanced TAT-Cre Protein Transduction for Efficient Gene Recombination in T cells.
Federica SgolastraChristina Arieta KuksinGabriela Gonzalez-PerezLisa M MinterGregory N TewPublished in: ACS applied bio materials (2018)
Genetic manipulation has increased our understanding of gene function and led to the discovery of new therapeutic targets. Cre/LoxP DNA recombination is widely used for genetic studies in mammalian cells. The direct delivery of Cre recombinase fused to protein transduction domains (PTDs), such as TAT, has been described as a valid alternative to the conditional, site-specific Cre expression in transgenic mice. However, efficiently conveying proteins into live cells, especially primary T cells, remains a major challenge. In this study, we show that one of our recently developed PTDs synthetic mimic greatly enhances the cellular uptake of the TAT-Cre fusion protein, enabling significantly smaller amounts of the protein to be used. We used this technique in primary mouse T cells to successfully delete, ex vivo, two essential genes involved in regulating T cell activation, Notch1 and Rbpjκ . Ex vivo gene deletion resulted in substantial protein reduction, comparable to that obtained in vivo when Cre-expressing Notch1 -floxed ( MxCre ± Notch1 fl/fl ) mice were treated with polyinosinic-polycytidylic acid (polyl/C), but in considerably less time, and without altering normal cell physiology. These results highlight several key advantages that include the ability to use less expensive protein (TAT-Cre), a major reduction in total experimental time and labor, and fewer side effects on the treated cells. This method should offer new opportunities for immunological studies, especially in the context of identifying novel therapeutic targets.
Keyphrases
- genome wide
- copy number
- protein protein
- induced apoptosis
- amino acid
- binding protein
- cell proliferation
- dna damage
- poor prognosis
- small molecule
- cell cycle arrest
- dna repair
- adipose tissue
- type diabetes
- high throughput
- dna methylation
- oxidative stress
- metabolic syndrome
- stem cells
- bone marrow
- skeletal muscle
- cell free
- endoplasmic reticulum stress
- cell therapy
- mesenchymal stem cells