Lab Protocols and Reagents

The following protocols are commonly utilized in our lab.

Protocols

In Vivo Experimentation

• Targeted genetic manipulation of mice
• Breeding and genotyping mice with various mutations
• Genetic, dietary and chemical models of obesity, diabetes and atherosclerosis
• Metabolic characterization of mice, indirect calorimetry, DEXA
• Clamp Studies
• Athesclerosis Studies
• Isolation of primary cells and establishment of cell lines

Cell Culture

• Thawing, propagating and freezing cell lines
• Serum preparations for special applications
• Differentiation of pre-adipocyte cell lines
• Cholesterol uptake and efflux
• Insulin signaling in differentiated adipocytes
• Deoxy-glucose uptake in preadipocytes and in differentiated adipocytes
• Stimulated lipoysis in differentiated adipocytes
• Co-culture protocols for differentiated adipocytes and other cells
• Metabolic labeling (³⁵S – ³²P)
• Immunocytochemistry

Protein Biochemistry

• Protein isolation from whole tissues and cultured cells
• Subcellular fractionation of tissue samples and cells
• Recombinant protein production, quantitation (Bacterial, Mammalian)
• Fusion proteins
• Affinity chromatography
• SDS-PAGE
• Immunoblotting, immunoprecipitaion
• In vitro kinase assays

Molecular Biology

• Total RNA isolation from whole tissues and cultured cells
• DNA isolation from whole tissues and cultured cells
• Northern-blotting
• Southern blotting, RT-PCR
• Mutagenesis
• Subcloning (vectors, enzymes)
• Ligation (blunt-end, sticky-end)
• Sequence analysis
• Agarose gel electrophoresis (regular, high resolution)
• Transient transfection
• Retroviral transfection/infection
• Adenoviral gene transfer
• Lentiviral gene transfer
• Luciferase reporter assays
• Mobility shift assays, DNAseI footprinting
• RNAi mediated gene regulation

Reagent Collection

The collection of biological reagents available in our lab include the following:

Vectors and cDNA clones

Expression/genomic libraries

• cDNA libraries from white and brown adipocytes
• Yeast-2 hybrid library from white adipose tissue

Cell lines

• Murine pre-adipocytes(e.g. 3T3-L1,F442A,TNFR1^-/-,TNFR2^-/-, TNFR1^-/-R2^-/-, aP2^-/-, mal1^-/-)
• Packaging cells (e.g. Bosc) and many more
• Monocyte/macrophage (RAW, U937, THP-1, aP2^-/-, mal1^-/-, LXR^-/-, PPAR^-/-)
• Primary macrophages (splenic/bone marrow/thioglycollate ellicited)
• Primary and established MEF cells from various mutants
• Antibodies

Murine mutant models

• Various Cre Models
• GATA2-LoxP
• GATA3-LoxP
• GATA2/3-LoxP
• STAMP1^-/-
• STAMP2^-/-
• TNF alpha^-/-, ob/ob & TNF alpha^-/-
• TNFR1^-/-, TNFR2^-/-, TNFR1^-/-R2^-/- and all in ob/ob background
• JNK1^-/-, JNK2^-/-, JNK3^-/-, XBP1^-/+
• aP2^-/-, mal1^-/-, aP2-mal1^-/- and all in ob/ob and ApoE^-/- backgrounds
• ob/ob, db/db
• yellow, tubby