Lentiviral Vectors Encoding GABAergic Enzymes

At a Glance

The lentiviral constructs used for neuronal reprogramming of human ES cells included Ngn2-t2A-PuromycinResistance (Tet-on promoter) and rtTA (Ubiquitin promoter), with an optional virus encoding EGFP (Tet-on promoter) for morphological analyses31. The cDNAs encoding human vGAT, GAD65, and GAD67 (V57 factors) were cloned into lentiviral vector driven by human Synapsin-1 promoter (see Supplementary Fig. S8b), followed by a Woodchuck Regulatory Element (WRE), and flanked by 5’ and 3’ long terminal repeats (LTRs).

Background

Glutamate decarboxylase or glutamic acid decarboxylase (GAD) is an enzyme identified as a major autoantigen in insulin-dependent diabetes. It is expressed in the brain as well as the insulin-producing β-cells of the pancreas. Its role is to catalyze the decarboxylation of glutamate to gamma-aminobutyric acid (GABA) and carbon dioxide (CO2). There are two isoforms of GAD that are present in mammals: GAD67 and GAD65. These are encoded by two genes, GAD1 and GAD2, respectively, located on different chromosomes (chromosomes 2 and 10 in humans). GAD1 may play a role in the stiff man syndrome, as well as other areas of pathology (such as autism, Parkinson’s disease, schizophrenia and bipolar disorders, cerebellar disorders and more). Its deficiency has been shown to lead to pyridoxine dependency with seizures.

The vesicular GABA transporter (VGAT) protein, also known as vesicular inhibitory amino acid transporter, is a member of amino acid/polyamine transporter family II. This transmembrane protein is involved in transporting gamma-aminobutyric acid (GABA) and glycine into synaptic vesicles.

Benefits

  • A method to induce GABAergic output synapses in glutamatergic neurons.

Applications

  • Ectopic expression of exogenous presynaptic enzymes and vesicular transporters
  • Expression of GABA for in vitro research
Last Updated: June 2023
Imaging of neurons
Inventors

Soham Chanda

Reference Number
2022-072
Licensing Manager

Steve Foster
Steve.Foster@colostate.edu
970-491-7100