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.

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.

Table 3 The transgenic studies using the SLC2A4 mini gene and its promoter for the whole-body expression in mice

Transgenic constructs	Analysis	Observations	Ref.
A 11.5-kb mini gene of human SLC2A4 starts with a 5.3-kb fragment upstream of transcription start and terminates within exon 10 of the gene followed by the bacterial CAT in pHSS6 vector	RNase protection assay and Western blot were used for SLC2A4 mRNA and GLUT4 protein in BAT, WAT, heart and skeleton muscle, respectively	The transgene expression was detected in WAT and BAT, heart and skeleton muscle of mice. Female transgenic mice have higher GLUT4 protein in the adipose tissue and less SLC2A4 mRNA in skeleton muscle than male ones. Transgenic mice have higher GLUT4 protein level in adipose tissue, liver, heart and skeleton muscle than the controls	[43]
The 11.5-kb minigene with the CAT reporter as shown in[43]	Reverse transcription PCR was used to measure SLC2A4 mRNA in cardiac and hindquarter muscle, BAT and WAT. Immunofluorescent test was for GLUT4 translocation	Transgenic mice gained more weight after 15 wk old of age, and have lower blood glucose in both fasting and fed states, lower insulin level in fasting and higher after refeeding, and higher glycogen contents, GLUT4 translocation in cardiac and skeleton muscle than the control mice	[44]
The 11.5 kb minigene with the CAT reporter as shown in[43]	Western blot was used to detect GLUT4 in gastrocnemius muscles	Transgenic mice have lower serum glucose level in both fasting and fed state, higher insulin level during fasting and lower after fed than the control ones	[45]
The 11.5 kb minigene with the CAT reporter as shown in[43]	Western blot was used to detect GLUT4 in the heart	Transgenic mice have higher glucose uptake, glycolysis and glycogen content, and lower insulin-stimulated glycolysis rate and glycogen synthesis in the heart than the control ones. Glucose and fatty acid oxidation remain the same	[46]
The 11.5 kb minigene with the CAT reporter as shown in[43]	Immunofluorescence was used to detect GLUT4 in cardiac myocytes and adipocytes	Transgenic mice have similar body weight, and epididymal adipose tissue weight and adipocyte size as the controls. Transgenic mice have higher levels of triglycerides, β-hydroxybutyrate and free fatty acids, and parametrial fat weight and lower glucose level after an oral glucose challenge and insulin level after an insulin injection than the controls. The insulin-stimulated glucose uptake is impaired in transgenic mice	[47]
A 2.4-kb of 5’ flanking DNA fragment of human SLC2A4 promoter fused with the CAT as a reporter construct	CAT activity assay and RNase protection assay were used to detect promoter activation and mRNA, respectively	In transgenic mice, CAT activity can be detected in the tissues that generally express GLUT4, including BAT and WAT, and smooth, skeleton and cardiac muscle, but not the liver	[42]
A 2.4-kb of 5’ flanking DNA of human SLC2A4 promoter fused to CAT as shown in[42]	Western blot was used to detect GLUT4 in adipose and skeleton muscle tissues	Transgenic mice have slower rise of blood glucose (no difference in glucose and insulin levels) during pentobarbital sodium anesthesia, and higher glucose infusion rate (40% increase) during hyper insulinemic euglycemic clamp than the controls	[48]
A 2.4-kb of 5 flanking DNA of human SLC2A4 promoter fused to CAT as shown in[42]	Only cited previous publications[42]	Transgenic mice have lower blood glucose, higher lactate and β-hydroxybutyrate levels during both fasting and fed states, and better glucose transport in the soleus muscle when fed a high-fat and high-sugar diet than the controls	[49]

BAT: Brown adipose tissue; CAT: Chloramphenicol acetyltransferase; GLUT4: Glucose transporter 4; Ref: References; WAT: White adipose tissue.

Table 4 Recombinant DNA techniques to create tissue specific glucose transporter 4 overexpression in animals and cells, analysis performed, and observations reported

Techniques	Tissue/analysis	Observations	Ref.
A 6.3-kb genomic DNA fragment of human SLC2A4 gene is under the control of a 5.4-kb’ DNA fragment of mouse ap2 promoter using Gateway cloning	Adipose-specific overexpression/ Western blot was used to detect GLUT4 in BAT and WAT	Transgenic mice have lower glucose level in the fasting, insulin level in the fed state, higher body weight and body fat at 18 to 21 wk of age, and higher basal and insulin-stimulated glucose transport rates in epididymal, parametrial, and subcutaneous adipocytes than the controls	[50]
Same as in[50]	Adipose-specific overexpression/Only cited previous publications[50]	Transgenic mice have higher body weight, parametrial fat pad weight and adipocyte size, and glucose transport in both fasting and fed states, and lower plasma insulin and glucose levels after a glucose challenge than the controls	[51]
Same as in[50]	Adipose-specific overexpression/Only cited previous publications[50]	Transgenic mice have higher glucose disposal rate in a glucose tolerance test, and palmitic acid-hydroxy stearic acid levels in serum, WAT and BAT than the controls	[52]
Same as in[50]	Adipose-specific overexpression/Western blot was used to detect GLUT4 in BAT and WAT	Transgenic mice fed a high-fat diet have higher glucose disposal rate than those fed a low-fat diet, and stable GLUT4 expression in fat and no increase in body fat	[53]
Same as in[50]	Adipose-specific overexpression/Western blot was used to detect GLUT4 in BAT and WAT	Transgenic mice have higher gonadal adipose weight, basal and maximum insulin stimulated glucose transport in isolated adipocytes, glucose transport rate, triglyceride synthesis and CO2 production than the controls	[54]
A 4.5-kb DNA fragment of the mouse Slc4a2 gene is under the control of a 3-kb fragment of the mouse myosin light chain gene promoter	Hindlimb muscle overexpression/Northern blot and Western blot were used to detect Slc4a2mRNA and GLUT4 in different tissues respectively	Transgenic mice have higher basal and insulin-stimulated glucose uptake and turnover, higher glycogen content in the skeleton muscle, higher insulin sensitivity, higher levels of free fatty acid and ketone in both fasting and fed state, and lower fasting glucose level than the controls	[55]
The human SLC2A4 cDNA is driven by the CMV promoter in pCIS2 vector	Rat adipocytes overexpression/Immunofluorescence was used to detect GLUT4 overexpression	Rat adipose cell transfected with the GLUT4 construct had significantly higher antibody binding after insulin stimulation than the control cells	[56]

BAT: Brown adipose tissue; CMV: Cytomegalovirus; GLUT4: Glucose transporter 4; Ref: References; WAT: White adipose tissue.