Looking for tips on cloning e coli for complete Myostatin knockout expirament in near future

I've been interested in Myostatin gene deletion for some time. e-coli is the carrier for the human Myostatin knockout Targeting crispr cas-9 plasmid I'll be using, however the dna concentration in the kit is not high enough. Dna replication can happen if I can grow a larger e-coli culture. Anyone know the exact procedure for growing e-coli in a controlled fashion? Once I grow a suitable amount of the bacteria I'll be using an endotoxin free plasmid dna isolation kit to isolate the human Myostatin knockout Targeting crispr cas-9 plasmid. Once isolated it will be combined with polyethylenimine in order to allow delivery into the cells. Any information on how to grow an e coli culture would be greatly appreciated. My observations results and any other questions I have will be posted here once I can begin this expirament.

Comments

  • Well growing bacterial cultures is rediculously easy. Are you sure there isn't something like a selective medium though being used to keep the change? Like does the medium you're working with have an antibiotic in it?
  • Yes I've heard it's incredibly easy yet can find no specific information on it which is strange to me. The medium to my knowledge does not have and antibiotic in it and isn't a selective medium. I believe it is just contains the Myostatin knockout Targeting crispr cas-9 plasmid being carried in an e-coli culture. Would I be able to simply grow the bacteria culture on an agar medium? And is there any way I would be able to measure out a specific amount of bacteria to use from the culture once I grow it?

  • If you grow in LB broth instead of agar you can you a spectrophotometer to measure the concentration of bacteria in the broth

  • That's promising! There wouldn't be any reason I would be unable to filter out the lb broth using an endotoxin free plasmid dna isolation kit once i measure the e-coli concentration, right?

  • edited February 7

    By the way the open human plasmid does have ampicillin resistance, so I would think the CRISPR-cas9 one does too. Maybe email the odin (assuming this is where you're getting your stuff).

    Honestly I don't think you'd need to measure the concentration of bacteria in the broth/media. You're probably going for the most you can get, so you go for a large dose. Then kit should filter out the lb broth if it's what I'm thinking of. You would also likely want to do multiple doses. One dose of even high concentration and large volume would likely not be as effective as you expect. You may need to grow a culture, take a small amount and put it in broth/plate, inject yourself with the purified plasmid from the original broth, and repeat.

    I would be very cautious with this. We now know that CRISPR seems to illicite an innate immune response. This may not be an issue for you on a physiological macro scale, however it may stop you from seeing any physiological changes post injection. There is also the chance of a severe allergic reaction to literally anything you inject into yourself. You would be wise to strongly consider this, what allergie you have, and whether you want an epipen/epinephrine and a partner.

    I too wanted to modify my myostatin gene, however the way the Dr. Zayner does it is very crude to me and potentially not as effective as other methods. I decided against doing this in myself and want to warn against others blindly following what Zayner did because it's cool. On the other hand I also want to help anyone who wants to go forward with this procedure after learning about it. If any of you do want help with the process I am willing to assist you.

  • Yeah, I definitely realize there is a large potential for allergic reaction and possible adverse effects. For this reason I plan on beginning with small doses to monitor for signs of allergic and adverse reaction and slowly ramping up my doses and dose frequency. I also see what you're saying about going for the largest doses possible and repeating the treatment. My plan is to grow the culture to have a larger abundance of it. The reason I want to be able to measure how much I have is to know how much polyethylenimine to mix in to achieve maximum effective delivery into the cells. What other methods do you think would be more effective than this method? What makes you view this method as crude?

  • @RealityWizard I'm very interested in any other methods for accomplishing this you have discovered. I haven't found any other methods other than this one and have basically dedicated all my research into this particular method.

  • @TylerEyth You could try a virus. There are again issues with that especially with the immune system, but from what I remember it is generally more effective. Of course a virus comes with all the issues of working with a virus especially if it's particularly virulent. From what I can tell this method would introduce less errors and mutations. You would have to research this in particular, but it should be easy enough to get the genome of some adenovirus that's used for gene therapy and insert your own sequence that should be integrated into the host genome. I suppose this may be overcomplicating things.

    There are also things like zinc finger nucleases and TALENS, but these are not as good as viral if I recall properly and they would take quite a bit of work. I'm also not sure you can reasonably use this in yourself.

    From what I know your best bet is a virus or CRISPR. If you do CRISPR there is some documentation on it from Zayner, but I would encourage you to do research yourself. There is information on how to get CRISPR to target more effectively one spot. You may also want to make it so that the gene change is not a knock-out, but rather a nucleotide change that indicates decreased or loss of function. With the virus you should look into previous gene therapy trials and see how they performed it. there should be substantial material on this. No matter what there's a lot of reading to do. I suggest thinking of your goal in the situation and strongly considering why and how to make the change.

  • @RealityWizard my plan is basically copying Zayner's experiment as closely as I can, monitoring for adverse effects, and continuing to do treatments in sites all over the entire body and slowly upping dosage and dosage frequency and monitoring for visual changes in muscular structure as well as changes in muscle power output. I am trying to figure out a practical way to use systems in my body to disperse the CRISPR to avoid having to do site specific injections all over my entire body. Would a virus be able to accomplish this?

  • @TylerEyth Admittedly I do not know for sure. CRISPR is still being researched, so it may be hard to compare. However the efficiency of the virus to enter your cells would be much higher than the pure DNA+chemical that you would likely use for CRISPR.

    I would avoid doing the whole body immediately. Start with small does on the forearms or calves. Somewhere well removed from the heart and brain. I would even consider using electroporation and possibly restricting blood flow with surgical tubing for a minute after injection. The reason behind this is to avoid any possible side effects. If you find yourself responding well to the treatment then go for more of the body.

    Here is a good paper on viral vectors for gene therapy if you want to read it, but it may not help a lot (https://doi.org/10.1016/S0163-7258(98)00020-5).

    I guess the easiest option is just using CRISPR and following Zayner. You may be able to improve the cas being used or use a different one entirely. Potentially one that has lower efficiency, but higher selectivity. I know in his page on the myostatin knockout he talks a little about his choice.

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