Lentiviral-Mediated shRNA Approaches: Applications in Cellular Differentiation and Autophagy

Orfali, Nina; Jeyapalan, Jennie N.; Woodcock, Corinne L.; O�Donovan, Tracey R.; Benjamin, Dalyia; Cahill, Mary; McKenna, Sharon; Gudas, Lorraine J.; Mongan, Nigel P.

Abstract

Acute myeloid leukemia (AML) is characterized by the accumulation of immature white blood cell precursors in the bone marrow and peripheral circulation. In essence, leukemic cells fail to differentiate and are stalled at a particular step of hematopoietic maturation and are unable to complete their development into functional blood cells with a finite life cycle. They are thus said to possess a “differentiation block.” Pharmacological override of this block is one attractive avenue of therapy, termed “differentiation therapy.” The most successful example of this therapeutic strategy is the use of the physiologic retinoid all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL). In this chapter, we will outline the methods used to characterize the mechanisms mobilized by retinoid signaling and will use the activation of a key regulator of autophagy, ATG7, as an example of the functional characterization of a retinoid regulated gene during differentiation. We will discuss how lentiviral delivery of shRNA constructs into cultured APL cells, such as NB4, can be used to functionally deplete key proteins. We will also describe how the effect of protein knockdown on ATRA-induced differentiation and autophagy can be assessed using quantitative PCR, Western blotting, and flow cytometry.

Citation

Orfali, N., Jeyapalan, J. N., Woodcock, C. L., O’Donovan, T. R., Benjamin, D., Cahill, M., McKenna, S., Gudas, L. J., & Mongan, N. P. (2019). Lentiviral-Mediated shRNA Approaches: Applications in Cellular Differentiation and Autophagy. In Retinoid and Rexinoid Signaling (33-49). Humana Press. https://doi.org/10.1007/978-1-4939-9585-1_3

• Online Publication Date: Jul 30, 2019
• Publication Date: Jul 30, 2019
• Deposit Date: Dec 11, 2019
• Pages: 33-49
• Series Title: Methods in Molecular Biology
• Series Number: 2019
• Book Title: Retinoid and Rexinoid Signaling
• ISBN: 978149399584
• DOI: https://doi.org/10.1007/978-1-4939-9585-1_3
• Public URL: https://nottingham-repository.worktribe.com/output/3535492
• Publisher URL: https://link.springer.com/protocol/10.1007%2F978-1-4939-9585-1_3
• Contract Date: Jul 30, 2019