Entry #5 Cait Barrett Sorting eggs

This week is mainly about sorting and collecting fish. These fish have been injected with a CRISPR protein that has a target site at the beginning of a gene called NOG and at the end. Our goal is to hopefully remove the whole gene from the fish’s genome. So far this has been a very unsuccessful gene for Ben, as no mutants have been found over the past year. This time we attempted a new target site during injection, as well as increasing the amount we were injecting the fish with. After we injected we allowed the fish to sit in the 55 degree Celsius incubator for about 3 hours in order for the eggs to begin to develop. When we looked at the eggs at 3 hours post fertilization we were just checking to see infertile vs fertile eggs, in order to discard the infertile eggs and also get infertility ratios of the parents. After this we allowed for the eggs to incubate overnight in the 45 degree Celsius incubator. Looking at them 24 hours post fertilization, we hoped to see patterns of ventralization. Ventralizing is when you begin to see more tail than head. There are different levels of ventralization, from wild type which has none to V5 which is an embryo with just a tail crumpled on the yolk. The more seriously ventralized fish won’t survive. We sampled some of these fish taking about 6 of each phenotype from the different clutches, and then performed HRM on them. The HRM results we got back showed that we had indeed made mutants this time around. This was a big step for Ben’s project, as he could not create mutants the previous year. In order to celebrate this we moved Ben’s dial on the lab struggle-meter down a notch.  

Along with sorting these CRIPR Nog fish, Ben and I also sorted embryos that we had injected with 3 different morpholinos for Woods Hole. These injections went very well as with each different morpholinos, mSmad5 a morpholinos that is normally used in mice and disrupts the Smad protein, xbmp7 which is found in xenopus and knocks out the Bmp7 receptor, and Chordin which is a different protein that has to do with ventral formation. For the mSmad5 and xBmp7 we saw dorsalization phenotypes. Dorsalization is when the tail becomes less prominent and these have a scale as well. From wild type to C5. With the Chordin morpholinos we saw a ventralized phenotype as we expected. In all this week has been pretty successful in terms of experiments so far. 

This picture is a chart that shows the different dorsalized levels, and the tail features that identify them from wild type to C5. 

   

This picture is a chart that shows the different ventralized levels, and the tail features that identify them from wild type to V5.