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

Table 1 Methods used to create whole body and tissue specific Slc2A4 knockout animals, tissues and animals studied, analytic methods included, and observations reported

Methods	Tissues/Animals	Analysis	Observations	Ref.
A construct with a disrupted mouse Slc2a4 gene was electroporated into WW6/22 ES cells to create deletion, which were microinjected into C57Bl/6 blastocysts	Skeletal muscle/GLUT4-null mice and wild-type control mice	Southern blot for DNA, Northern blot for mRNA, and Western blot for protein measurements	The Slc2a4-/-mice have normal glycemia, growth retardation, decreased longevity, cardiac hypertrophy, reduced adipose deposits, postprandial hyperinsulinemia, and lowered insulin sensitivity; The male Slc2a4-/-mice have lower and higher blood glucose levels than the controls in fasted and fed states, respectively	[31]
GLUT4-loxP mice were crossed with α-MHC promoter-driven Cre	Heart/Cardiac-selective Slc2a4-/-deletion mice (G4H–/– mice) and control mice	Southern blotting and PCR for DNA, and Western blot for GLUT4 levels using various antisera	G4H–/– mice have modest cardiac hypertrophy, normal life span and serum levels of insulin, glucose, FFAs, lactate, and β-hydroxybutyrate, increased basal cardiac glucose transport and GLUT1 expression, and abolished insulin-stimulated cardiac glucose uptake	[33]
GLUT4loxP mice as shown in[33] were crossed with the muscle CK promoter driven Cre transgenic mice to obtain Muscle-G4KO	Skeletal muscle/Muscle-G4KO mice and heterozygous Slc2a4 deletion mice in the 129SV and C57Bl/6J background	Reverse transcription–PCR for mRNA, and Western blot for GLUT4 protein (anti-GLUT4 AB1346)	Muscle-G4KO mice show a reduction in basal and near-absence of insulin- or contraction-stimulated glucose transport, showing; severe insulin resistance and glucose intolerance from an early age	[34]
GLUT4-null mice were crossed with transgenic mice expressing GLUT 4 driven by MLC promoter[55] to create MLC-GLUT4-null mice	EDL and soleus muscle/MLC-GLUT4-null mice having GLUT4 in the fast-twitch EDL muscle, GLUT4 null mice, and control mice	Western blot for GLUT4 protein (rabbit polyclonal antiserum)	MLC-GLUT4-null mice have less GLUT4 in WAT (females only) and soleus muscle, adipose tissue deposits, adipocyte size, and plasma free fatty acid levels in the fed state than the controls. Glucose uptake in the EDL, but not in the soleus, muscle is restored to normal in male and above normal in female MLC-GLUT4-null mice	[32]
GLUT4–loxP mice were crossed with aP2-driven Cre transgenic mic to obtain G4A-/- mice	Adipose tissue/G4A-/-, and control mice	Western blot for GLUT4 protein in BAT and WAT tissues	G4A-/- mice show impaired insulin-stimulated glucose uptake in adipocytes, glucose intolerance, hyperinsulinemia, and insulin resistance in the muscle and liver	[35]
The G4A-/- mice[35] were crossed with the muscle-G4KO mice[34] to generate AMG4KO mice	Adipose tissue and skeletal muscle/G4A-/-, muscle-G4KO, and AMG4KO mice	Western blot for GLUT4 protein using antibodies from H. Haspel in the Charles River Laboratory	AMG4KO mice develop fasting hyperglycemia and glucose intolerance and are at risk for greater insulin resistance than mice lacking GLUT4 in only one tissue	[37]
The neuron-specific Nestin promoter-driven Cre transgenic mice were crossed with GLUT4-loxP mice (FVB strain) to obtainBG4KO mice	Whole brain/BG4KO and control mice	Western blot for GLUT4 protein in the brain using antibody from Chemicon	BG4KO mice have glucose intolerance, insulin resistance, and impaired glucose sensing, suggesting that the brain GLUT4 may sense and respond to glucose	[36]

α-MHC: α-myosin heavy-chain; AMG4KO: Adipose/muscle-GLUT4 double knockout; BAT: Brown adipose tissue; BG4KO: Brain-specific GLUT4 knockout; Cre: Cre recombinase; CK: Creatine kinase; ES: Embryo stem; EDL: Extensor digitorum longus; GLUT4: Glucose transporter 4; G4A-/-: Adipose tissue-specific GLUT4 knockout; GLUT4-loxP: Slc2a4 allele with exon 10 flanked by loxP sites; G4KO: GLUT4 knock out; MLC: Myosin light chain; Ref: References; WAT: White adipose tissue.