The first stage was to see if Uaas could be incorporated into neural stem cells, without disrupting their process of differentiation, and if so, would the fluorescent tag they inserted be carried into neuronal cells created by the stem cells.
"Current methods for Uaa incorporation are not appropriate for stem cells, because the added genes are often lost before the stem cell has a chance to finish differentiation," Wang says. To solve that problem, the researchers developed a lentiviral-based gene delivery method to incorporate the Uaas into proteins expressed in neural stem cells. "The lentiviral gene therapy technique, which was pioneered by Dr. Inder Verma at Salk, can afford long-lasting expression through stem cell differentiation," Wang says.
The virus was used to deliver different components needed in the Uaas technology. These consisted of a synthetic transfer RNA (tRNA) that cells use to incorporate amino acids into a protein that is being built inside a cell. The second molecule is an enzymatic synthetase that can recognize the engineered tRNA, and load it with the third engineered molecule - an unnatural amino acid. These amino acids are chemically distinct from the 20 amino acids that naturally exist in the body; they can be engineered for different desirable properties, such as to fluoresce.
"Once you have a stem cell line stably incorporating Uaas, you can custom the Uaa to study stem cell biology," Wang says. "You also get the bonus of obtaining various mature cells with this ability through differentiation, such as neurons, which are difficult to insert Uaas in and expensive to procure in large amounts."
In the first set of experiments, the researchers found out that Uaas were successfully incorporated into neural stem cells, the incorporation lasted through the differentiation, and these cells then produced neurons carrying the fluorescent amino acid.
The second set of exper
|Contact: Andy Hoang|