Lentiviral-Mediated shRNA Approaches: Applications in Cellular Differentiation and Autophagy. Article uri icon

Overview

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.

publication date

  • January 1, 2019

Research

keywords

  • Autophagy-Related Protein 7
  • Leukemia, Myeloid, Acute
  • RNA, Small Interfering
  • Tretinoin

Identity

Scopus Document Identifier

  • 85070550750

Digital Object Identifier (DOI)

  • 10.1007/978-1-4939-9585-1_3

PubMed ID

  • 31359387

Additional Document Info

volume

  • 2019