My mutants have phenotypes! · 3:49am Jul 3rd, 2018
I am very pleased. My hypothesis appears to be correct!
Deleting Bcat from the Lgr5-expressing cells in the nasopharngeal primodium results in a huge bilateral cleft palate by embryonic day 15.0. But, it would allow the mice to breathe, so this explains why I have some adult Bcat mutants
Deleting the Lgr5 from the Lgr5-expressing cells from this region results in choanal atresia and a slowly-fusing cleft palate with variable gaps.
Deleting BOTH Lgr5 and Bcat from the cells causes the most severe phenotype, completely sealing off the sinus cavity from the mouth and pharynx, leaving the sinuses a large blind cavity.
The soft palate is fused in various locations in the Lgr5 mutants, and interestingly, appears completely absent in the Bcat mutant by itself.
So, now I have proof that these Lgr5-expressing cells are crucial for the patterning of the upper and soft palate, the sinuses, and the nasopharynx. They utilize an Lgr5-mediated Wnt-Bcat signaling pathway as part of their control system.
Given that this choanal atresia phenotype is associated in 4 syndromes, each possessing mutations in FGFR2, and there is a craniofacial mesenchyme-specific isoform of this receptor expressed in the same regions as the Lgr5, I can deduce that these two pathways are working in tandem to properly develop the palate and nasopharynx. And indeed, from searching the literature, I have found that FGFR2 isoforms elsewhere repress another protein FOXL2, which suppresses SOX9... and SOX9 is often required for upregulating Lgr5! Therefore, the FGFR2 mutants may be upstream dysregulators of Lgr5-mediated Wnt-Bcat signaling in the craniofacial region!
I shall now take pictures of the mutant embryo sections and toss together my GoFundMe page over the weekend so I can keep the crucial mouse lines alive and afford the next set of experiments I must perform.
I congratulate you sir. Here's to hoping you stay in business a little bit longer *raises a shot glass*
4894007 It's not 'business', per se, so much as it is when the lab closes I'd have to euthanize the mouse colony, and it would take quite a long time and a lot of money to recreate the lines in another lab.
I can save the samples I've already taken, since they're just little frozen blocks in a few small freezer boxes, but I can't get any new samples if I lose the colony, and the odds that I have all the samples I need is pretty much lower than my odds of winning Powerball and Megamillions simultaneously. When exploring a signal transduction pathway during development, you go through an absurd number of samples trying to track down all the genes involved. Plus, you need ones of different fixation times, oriented in different directions to get different angles of cuts, some need to be in paraffin instead of frozen for certain experiments that work better with the very thin slices you can get with a paraffin microtome.
It's probably 10x cheaper just to get a GoFundMe and pay for the colony maintenance myself. $10K to keep the colony alive at a modest number of cages vs probably 100K when all the costs are accounted for to rebuild the entire colony from scratch, which would take a solid year just to generate the compound mutant lines again.
It takes about 3 months for a generation to be born and reach breeding age. With 8 transgenes (rko, r1a-flox, Bcat-flox-KO, Bcat-gain, r1bko, Rosa-flox, Lgr5-KO-Cre, TGFBR2-flox-KO) all from different founder lines to cross together into the various experimental lines, if you're REALLY lucky you can get the full complement in 3 generations. Then you have to breed up enough of those to perform experimental matings. Again, REALLY lucky you get enough after one more generation. And that's a year. Just buying the initial founder lines you need costs a couple thousand, and the breeding will greatly expand the colony size until you have enough of the compound mutants. Now, at about $2 per cage per day... which means 100 cages costs $200 PER DAY, or $6,000/month... that's $72,000 a year just to breed the mice you need; and you also have to genotype all those mice. The Taq polymerase I use costs nearly $400 for enough to do 200 PCR reactions, and each mouse needs 3 PCRs or more... one line has 5 transgenes in it... and I'd be doing that on a couple thousand mice, let's say 2,000 for starters. That's $12,000 just in Taq polymerase.). So, $84,000 as a base number JUST TO GET THE COLONY ESTABLISHED!! No experiments done during the entire period.
Science is not for the impatient.
Nice. Back in my university days I only found out my mutants could see something after getting a sodium iodate injection, while regular mice got blind as fuck (it was a complement-disabling mutation, so the immune system reaction to iodate didn't wreck the retina that much).
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*Nit-picking kicks in* Well, when I said "stay in business" I meant employed *nit-picking turns off*
I agree; 10K is a lot cheaper than 100K. I also understand that the mice are technically the property of your employer, but do they really just kill off all the mice just like that when they stop something? Can't they use them again for something else or sell them? (I think I just answered my own question while typing this, realizing that since the mice aren't "pure" stock, any findings in another study that uses them could be scewed, so I shall redact my question of reusing them)
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Isn't any benefits one gains from these experiments and advancements in the betterment of our species really just gravy on top the fact, right now, as your reading this, someone, who we can assume is taking a break from blogging about organic sourced coffee, put a shaking hand to their mouth, then turns to their ten cats and says in a horrified voice "these monsters are experimenting on mice!"
Right now Alondro, you're making a leftist cry.
4894105 It's rather up to us to find another use for the mice, or another lab. That's actually one disadvantage of working in non-profit research; there's not as much incentive to preserve things for the lead institution. A traditional for-profit company will be more loathe to throw away a significant investment. But, by the same token, such a company is also unlikely to fund research that has little chance of generating a profit even if totally successful.
There are always advantages and disadvantages to both.
I'm also doing some tissue work for a tiny fledgling pharmaceutical company. They have a drug that's immensely promising for treating a number of degenerative and metabolic diseases. It works by preventing cellular damage at the mitochondrial level.
4894177 I suspect none of them would volunteer in the mice's place.