ABSTRACT
The ability to efficiently modulate autophagy activity is paramount in the study of the field. Conventional broad-range autophagy inhibitors and genetic manipulation using RNA interference (RNAi), although widely used in autophagy research, are often limited in specificity or efficacy. In this chapter, we address the problems of conventional autophagy-modulating tools by exploring the use of three different CRISPR/Cas9 systems to abrogate autophagy in numerous human and mouse cell lines. The first system generates cell lines constitutively deleted of ATG5 or ATG7 whereas the second and third systems express a Tet-On inducible-Cas9 that enables regulated deletion of ATG5 or ATG7. We observed the efficiency of autophagy inhibition using the CRISPR/Cas9 strategy to surpass that of RNAi, and successfully generated cells with complete and sustained autophagy disruption through the CRISPR/Cas9 technology.
Subject(s)
Autophagy , CRISPR-Cas Systems , Gene Editing/methods , Animals , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 7/genetics , Cell Line , Cloning, Molecular/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Mice , RNA, Guide, Kinetoplastida/geneticsABSTRACT
To estimate energy expenditure (EE) in elderly subjects, more age-specific data are required on energy costs of standardized activities. EE was assessed by using indirect calorimetry in 28 women aged 72 +/- 4 y (mean +/- SD) and in 29 middle-aged women (42 +/- 1 y) at rest (resting metabolic rate; RMR) and during sitting, sitting with standardized arm activity, and walking on a treadmill at 3 km/h. RMR and EE during sitting, and sitting with standardized arm activity did not differ significantly between the groups, although EE expressed as a ratio of arm activity to RMR (physical activity ratio, PAR) tended to be higher in the elderly subjects. Walking EE was significantly higher in the elderly women (16.4 +/- 4.0 kJ/min) than in the middle-aged women (12.7 +/- 2.3 kJ/min), also when expressed as PAR. It is suggested that elderly women walk less efficiently. Because PARs are frequently used to estimate daily EE, it is important to note that additional age-specific data might be required.
