Optical Experiments Results
|Type of C. elegans||Fluorescence of mCherry / GFP||Fluorescence of GEM-GECO||CoChR work with GEM-GECO||CoChR work in behavioral Experiments|
|Odr10::CoChR::GEM-GECO::mCherry worms||mCherry is very bright and beautiful(Fig.1 & 2)||weak fluorescence in 497~527nm. Weak change after add diacetyl (Fig. 3).||Still testing||Successful.(See here)|
|Str1::Chrimson::GEM-GECO::GFP worms||GFP were observed in AWB neurons (Fig. 4)||weak fluorescence||Still testing||Exist response, but need more experiment to confirm.|
- mCherry expresses successfully in odr10::CoChR::GEM-GECO::mCherry worm in AWA neurons. The 3D video was captured by Luxendo Light-Sheet Microscope.
- Emission change at 497~527nm of GEM-GECO after adding diacetyl. It maybe was positive result, but need more control experiments to confirm it.
- GFP expresses successfully at AWB in str1::Chrimson::GEM-GECO::GFP worm.
Here, we fixed the Caenorhabditis elegans in the Immobilization Chip to observe the Odr10::CoChR::GEM-GECO::mCherry worms under the fluorescence microscope and saw the neuronal activity successfully, which can confirm that the worms can express our target genes. We also put the Odr10::CoChR::GEM-GECO::mCherry worms into the chip, after a few minutes the worms would be inactive, then we can "wake up" the worms by the blue light.
Then, we demonstrated that the insertion did not damage the olfactory receptor neuron pairs of the worms by testing their response to diacetyl and 2-nonanone in the Gaussian Plate.
Here, we confirmed that the Odr10::CoChR::GEM-GECO::mCherry worms could sense the blue light by inducing the Odr10::CoChR::GEM-GECO::mCherry worms to crawl a cycle on NGM plate. The Odr10::CoChR::GEM-GECO::mCherry worms could follow the blue light spot just like the attract of the food.
Then, in order to study the worms' learning ability we put the worms in alcohol layer on the NGM plate and stimulated them by the blue light at the same time. After 2 hours' training we found that the worms could crawl towards to the alcohol.
Plasmid Construction Results
According to the design of plasmid construction, we constructed Odr-10::CoCHR::GEM-geco::mCherry and str-1::Chrimson::GEM-GECO::GFP fusion genes in backbone pCFJ909 successfully. The fusion gene segments were all be sequenced.
We also amplify B series plasmid in miniMos system for microinjection. We integrated Odr-10::CoChR::GEM-GECO::mCherry and str-1::Chrimson::GEM-GECO::GFP fusion genes into C. elegans(Caenorhabditis elegans) by microinjection respectively.
In order to get C. elegans strains with the preference to blue lights and the aversion to red lights, we used miniMos injection to inject our plasmids in to worms for expression.
On July.7, we microinjected 20 worms with Odr-10::CoChR::GEM-GECO::mCherry and 20 worms with Str-1::Chrimson::GEM-GECO::GFP. After 3 days, for each kinds of worms, we obtained more than 6 free-moving F1 with fluorescences. Then, 10 days after microinjection, we did heat shock to screen stable inheritance worms. For worms with Odr-10::CoChR::GEM-GECO::mCherry, one plate successfully survived more than 30 worms without GFP(a selective marker), but none of worms with Str-1::Chrimson::GEM-GECO::GFP survived. In addition, we did mapping experiments and demonstrated that Odr-10::CoChR::GEM-GECO::mCherry had successfully inserted in chromosome 1.
On August 1st, we microinject worms using Str-1::Chrimson::GEM-GECO::GFP. This time we injected 20 worms and also observed F1 phenotype after 3 days, picking up free-moving worms with RFP and did heat shock 9 days later. After heat shock, on August 21th, we got about 10 worms expressed plasmids without arrays, meaning that we obtained stable inheritance worms expressing Str-1::Chrimson::GEM-GECO::GFP.