Uses of knockout, knockdown, and transgenic models in the studies of glucose transporter 4

Table 2 Methods used to knockdown glucose transporter 4 and analyze its expression in cell lines, and reported observations

Methods	Cells	Analysis	Observations	Ref.
Recombinant lentivirus was used to express shRNA based on SLC2A4 sequence (NM_001042.3)	Human head and neck squamous cancer cell lines, HSC-2	Western blot for GLUT4 protein using antibody from Epitomics	The knockdown of GLUT4 expression in HSC-2 cells induced DDX58 and OASL protein expressions, and reduced cell migration in culture	[39]
pSIREN RetroQ system was used to obtain recombinant retroviruses that produce shRNAs corresponding to mouse Slc2a4 sequence GGTGATTGAACAGAGCTAC (GenBank ID was not provided)	3T3-L1 adipocytes	Immunofluorescence of phase-contrast and epifluorescence images for GLUT4 protein using antibodies (rabbit anti-GLUT4, a gift from Dr. Sam Cushman (National Institutes of Health)	GLUT4 knockdown does not affect IRAP trafficking, showing that IRAP traffics is independent of GLUT4	[40]
Recombinant lentivirus was used to generate shRNA under the control of human H1-RNA promoter using the mouse Slc2a4 mRNA sequence (GenBank ID not provided)	3T3-L1 adipocytes	Immunofluorescence microscopy and Western blot for GLUT4 using rabbit polyclonal antibody from Chemicon International Inc	GLUT4 knockdown in 3T3-L1 adipocytes reduces insulin-stimulated glucose uptake by 50%-60%, IRAP expression of depending on differentiation stage, and lipogenic capacity of differentiated, but not differentiating cells	[41]

DDX58: DExD/H-Box Helicase 58; GLUT4: Glucose transporter 4; IRAP: Insulin-regulated aminopeptidase; OASL: 2’-5’-Oligoadenylate Synthetase Like; Ref: References; shRNA: Short hairpin RNA.