Exam 2 Optopatch Flashcards
What are the clinical and scientific significances of optopatch?
Clinical- use it on tissues from biopsies or neurons from stem cells
Scientific- less invasive method for exciting and recording from identified neurons, record from multiple neurons at the same time, get subcellular information with the use of GEVI
Shortcomings of GECI and GEVI
GECI- Hard to record cells that fire rapidly or buffer lots of calcium.
GEVI- Spectral overlap and the same wavelengths excite both the sensor and actuator.
Discuss the optopatch construct: Quasar2, dark morange CheRiff, EGFP
- Quasar2- sensor that is stimulated with long wavelength light.
- dark morange- used because it increases insertion into plasma membrane but does not fluoresce
- CheRiff- A type of channelrhodopsin, which is an activator, that is activated by blue wavelength light.
- EGFP- linked to CheRiff so CheRiff-EGFP fluoresces green.
Discuss the insertion of the optopatch construct into the targetting vector
It is inserted into the targetting vector downstream of the CAG promoter, and a STOP codon flanked by like oriented lox sites.
What is the ROSA26 locus and why is it optimal for the insertion of the transgene if you want to do a knockin?
The viral vector is targeted into intron x of the ROSA26 locus. ROSA26 is expressed in all cells, and less expression is in neurons. Targeting intron x does not decrease ROSA26 protein production or interfere with cell health.
How do you determine the presence of the optopatch construct?
G1/S12 primers flank part of the stop codon and part of the optopatch. They only recognize the optopatch so their PCR product will tell you if you successfully knocking in the gene. It does not show a band for the wildtype.
How do you determine the genotype of the optopatch construct?
The GT5/GT8 primers flank the wildtype, so they only recognize the wildtype. If there is a band at the wildtype, there should also be a band at the loxp/wt so you know that the mouse is heterozygous for the wildtype. If there is no band at the wildtype, then you know that the mouse is homozygous for the optopatch.
If the GT8/GT5 recognize the sequences that are present in both the wildtype and the knockin, then why is there not a band?
The knockin adds much more bases than the PCR is capable of amplifying.
What did the researchers conclude when they expressed cre that was driven by various promoters/enhancers?
They concluded that the expression of cre depends on the promoters and enhancers that express it.
Why is the peripheral nervous system more readily accessible for optopatch?
It does not have local circuit neurons.
Does the staining of the somatostatin overlap with the staining of the SST-Cre mouse and how did they find out?
Yes they saw co-expression of the SST and the SST/Cre. They used an antibody against the SST in the SST/cre mice. They saw the green fluorescence from the GFP and both overlapped.
How did they assess their negative controls?
- SST/Cre mouse combined with mouse with two copies of floxopatch. Showed bright labeling.
- Cre only mouse was combined with mouse with two copies of the floxopatch. Saw very little labeling.
How did the researchers conclude if floxopatch effects mice health?
they looked at the weight of the mice with floxopatch and two different cre drivers and the weight did not change so the animals were healthy.
Does optopatch effect the electrophysiology of neurons?
No because they saw no change in membrane resistance, capacitance, membrane potential or action potential threshold.
The researchers concluded that Quasar2 (GEVI) is slow to record the fast spiking neurons but how did they quantify it?
They looked at the full width at half maximal between the baseline and the height of the voltage recorded by the whole cell patch recording. Then they did the same for the fluorescence (Quasar) and found that the half maximal width was lower and wider.
