TAAR1 Expression Trial March 12-17, 2017

TAAR1 Expression Trial March 12-17, 2017

Week of 03/12-03/17 Experiment Summary

TAAR1 Expression Experiments

Purpose: Study differential expression of TAAR1 pGHE and pcDNA3 to determine the cause of recent expression problems with TAAR1. T271C pGHE from the poster was also injected to attempt to replicate poster data.

TAAR1 pGHE Seq Verification

We sequenced the entire TAAR1 ORF to reveal the 5’ and 3’ Xenopus globin UTRs. The sequenced aligned perfectly with the electronic sequence on file. Below are pictures of 5’ and 3’ UTRs adjacent to TAAR1 proving its presence in pGHE. The results below are from 12/2016.

In vitro transcription

wt TAAR1 pcDNA3, pGHE, and T271C pGHE were linearized in transcribed. Following cleanup and resuspension in 6uL of nuclease-free water, they were analyzed via gel electrophoresis. 1uL of each RNA sample was loaded. A picture of the gel can be found below.

RNA Microinjection

Following verification, TAAR1 RNA samples were diluted to 20% and 30% in separate tubes. All RNA tubes were complemented with 0.1%CFTR and the final volume was 10uL. Oocytes were injected with the 30% TAAR1 mixtures for expression tests. Injections occurred on 03/10/17. Mario injected both wt TAAR1 RNAs, and Shanna injected T271C.

TEVC Experiments

Experiments were done on the third, fifth, and sixth day after injection. Around the third day after injection, oocytes began to die, making it difficult to collect good data. All attempts were made good oocytes were picked for experiments.