--- Log opened Sat Mar 24 00:00:27 2018
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05:44 < kanzure> hmph
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07:28 < kanzure> "CRISPR based tools can be further modified with either meiotic specific expression, or fusion with a meiotic protein as an effector, to induce meiotic recombination at a specific target site and potentially increase genetic and epigenetic variations of favorable traits that involve linked alleles such as clustered plant immunity genes (Deng et al., 2017)."
07:29 < kanzure> ref is: Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance, Deng 2017
07:29 < kanzure> mutations to increase meiotic chromosome recombination crossover events (in plants at least) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712510/
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08:22 < kanzure> "A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells" https://academic.oup.com/nar/article/31/11/2952/1220264
08:23 < kanzure> oh they mean homologous recombination... not chromosome crossover recombination.
08:24 < kanzure> is there really no protocol yet for in vitro chromosomal crossover recombination?
08:41 < kanzure> hmm looks like meiotic recombination is a large source of errors in human reproduction (either due to chromosomal abnormalities or errors with crossovers or chromosomal arrangement)
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09:07 < kanzure> meiotic recombination should be separated from oocyte fertilization (which could be replaced with intraspecies/xenogenic ooplasm or nuclear transfer injection into oocytes, or electrofusion or other cell fusion methods, followed by delivery of oocyte activation factors) if there was a way to skip the recombination step of meiosis
09:08 < kanzure> with uniquely fluorescently-labeled chromosomes maybe it would be possible to use micromanipulation to manually force the separation of specific chromosomes into different cells, as a physical intervention to meiosis
09:09 < kanzure> direct recombination of human donor chromosomes is possible but i worry that it would lose epigenetic state or methylation or something, especially if you start processing the dna in vitro
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09:12 < kanzure> if epigenetic state was not a concern then you would basically want to manually pick and choose chromosome fragments and copy those into new chromosomes to produce the offspring genome, rather than relying on nearly-random crossover events
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09:16 < kanzure> probably meiotic recombination was studied 40 years ago and so that's why none of this stuff looks interesting (recency bias in research)
09:38 < kanzure> magnetic motion control and movement of chromosomes in oocytes http://s-space.snu.ac.kr/bitstream/10371/118542/1/000000136035.pdf using bacterial magnetic nanoparticles conjugated to "oocyte-specific linker histone H1 protein" (also the nanoparticles were fluorescent too?)
09:41 < kanzure> in that paper, they also propose using magnetic nanoparticles to physically move chromosomes as a way to "enucleate" the oocyte during somatic cell nuclear transfer, without removing meiosis-specific factors usually removed during spindle removal in typical physical enucleation
09:42 < kanzure> not really sure why they didn't try to push chromosomes out.. or why micromanipulation isn't used for this.
09:56 < kanzure> .wik whole chromosome painting probe
09:56 < yoleaux> kanzure: Sorry, that command (.wik) crashed.
09:57 < kanzure> ok looks like basic FISH stuff works (fluorescence in situ hybridization)
10:03 < kanzure> like here http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180158
10:03 < TMA> .title
10:03 < yoleaux> Pooling strategy and chromosome painting characterize a living zebroid for the first time
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10:09 < kanzure> geeze chromosome micromanipulation studies were done during 1940-1960s. this would explain lack of good recent science around this. probably forgotten stuff.
10:12 < kanzure> mostly kinetochore studies
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10:32 < kanzure> .wik micrurgy
10:32 < yoleaux> kanzure: Sorry, that command (.wik) crashed.
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10:41 < kanzure> here is a study where someone moved chromosomes between cells in different phases of meiosis https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2150703/
10:41 < kanzure> "We used a micromanipulation needle to fuse grasshopper spermatocytes in meiosis I to spermatocytes in meiosis II, and to move chromosomes from one spindle to the other. Chromosomes placed on spindles of a different meiotic division always behaved as they would have on their native spindle; e.g., a meiosis I chromosome attached to a meiosis II spindle in its normal fashion and sister ...
10:41 < kanzure> ...chromatids moved together to the same spindle pole. We also showed that meiosis I chromosomes become competent meiosis II chromosomes in anaphase of meiosis I, but not before. The patterns for attachment to the spindle and regulation of cohesion are built into the chromosome itself. "
10:43 < kanzure> "meiotic apparatus transfer (spindle transfer) between mouse oocytes" https://doi.org/10.1017/S0967199401001022
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12:54 < nmz787> hmm, so just enucleate and renucleate after meiosis has occurred?
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13:37 < kanzure> yeah i was wrong about that, sorry, basic biology fail.
13:38 < nmz787> hmm? I thought you were quoting articles
13:43 < kanzure> there's no meiotic recombination in fertilized eggs
13:44 < kanzure> and besides, recombination isn't important to viability, just to evolution
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13:49 < kanzure> page 2 figure 1 is pretty neat: http://diyhpl.us/~bryan/papers2/bio/in-vitro-fertilization/Xenografting%20of%20testicular%20tissue%20pieces:%2012%20years%20of%20an%20in%20vivo%20spermatogenesis%20system%20-%202014.pdf
13:50 < nmz787> kanzure: why were you saying intranuclear injection efficiency was too low? this paper seems to indicate 90%
13:50 < nmz787> .title http://www.nrcresearchpress.com/doi/abs/10.1139/b86-173#.Wra38R_OC00
13:50 < yoleaux> An Error Occurred Setting Your User Cookie
13:50 < nmz787> A detailed procedure for the intranuclear microinjection of plant protoplasts
13:50 < nmz787> 65k results on google scholar for intranuclear injection :/
13:53 < nmz787> "Microinjection rates of 80- 100 nuclei/h were routine. The delivery of volumes as small as 10^-17 L was easily controlled"
13:55 < nmz787> "The volumes injected in the above studies ranged from 10^-10' to 10^-13 L, which is at least 50 times greater than the volume of an alfalfa nucleus"  (speaking of previous work they're referencing)
13:57 < nmz787> hmmm, not sure what this is about "A small drop of mercury was loaded from the back of the pipette and advanced forward (Kiehart 1982)."
13:58 < nmz787> "Fine control was achieved through a pressure -volume transducer as described by Kiehart (1982). The system permits the operator to start and stop the flow of liquids into the nucleus without generating back pressure that could result in plugged pipettes."
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13:59 < nmz787> welp, they don't even mention the size or shape of the pipette tips... but they're just simply pulled glass capillary
14:00 < nmz787> how do we stimulate biohackers to do micro/nano injection? I can supply the glass pipette tips
14:01 < kanzure> 90% is okay but if you have another method in your chain with 3% success rate then you're suddenly screwed
14:01 < kanzure> and limited supply of oocytes means you don't get many chances
14:02 < nmz787> these folks want to charge $17 for each tip: https://www.wpiinc.com/products/laboratory-supplies/make-selection-pre-pulled-glass-pipettes-silanized/
14:02 < nmz787> 100nm to 10 microns inner-diameter (ID)
14:06 < nmz787> well what's the efficiency of gene-gun? that seems to be a curve as well, since we heard bigger DNAs get sheared during ballistic impact
14:12 < kanzure> that's probably fine- just use more cells. but when you're down to the single cell territory, things start to get.. weird.
14:12 < kanzure> "just make more DNA" doesn't work either, because in vitro replication of DNA doesn't necessarily preserve all of the epigenitic fluff
14:19 < nmz787> "just use more cells" seems to be directly against one of your last comments "limited supply of oocytes means you don't get many chances"
14:23 < kanzure> yeah but "use more cells" is the answer to low efficiency in biology-- that's literally how biologists make things work.
14:24 < kanzure> extraction, modification, and reimplantation of spermatogonial stem cells https://academic.oup.com/humupd/article/22/5/561/1749854   (but you have to irradiate the testis before you re-deliver otherwise mosaicism...)
14:26 < nmz787> what about an implant, similar to existing implantable birth control, which modifies eggs as they are dropped down naturally (so no risking surgical extraction, which scares women and their partners)
14:27 < kanzure> still limited transfection success will limit your outcomes there.
14:27 < kanzure> and also, the fabrication and construction of that device would be an immense challenge, compared to cutting up testicles and inserting them under mouse skin
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14:33 < nmz787> idk, DNA packed with lipofectamine in some months-long slow-release?
14:33 < nmz787> seems easy enough
14:33 < nmz787> partial success is still a partial win
14:33 < nmz787> if it's easy enough to use
14:35 < nmz787> heck, even some 'special lube' seems like it would be on the edge of being snake-oil... but if it provided /some/ chance of working... hrmm
14:35 < kanzure> spermatozoa have endonuclease activity
14:36 < nmz787> I honestly hope I don't see people geting ripped off like that in my lifetime
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16:12 < fenn> https://www.washingtonpost.com/news/the-switch/wp/2018/03/22/amazon-issued-patent-for-delivery-drones-that-can-react-to-screaming-flailing-arms/
16:13 < fenn> premium snake oil based lubricant
16:13 < kanzure> "Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells" https://www.sciencedirect.com/science/article/pii/S2329050117300359
16:13 < kanzure> "To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its ...
16:13 < kanzure> ...integration in native genome of mice. Such males generated transgenic progeny by natural mating."
16:16 < kanzure> hmm this is diybio level of skillz i think
16:18 < fenn> do they report on efficiency? quantitatively
16:19 < kanzure> "for this newly developed transgenesis technique, the overall efficiency to transmit a transgene in the first generation was found to be 46%"
16:20 < kanzure> lower than testi electroporation
16:21 < kanzure> but also doesn't require electroporation. so...
16:22 < fenn> wow that's really high
16:36 < kanzure> earlier version of the paper was on biorxiv https://www.biorxiv.org/content/biorxiv/early/2016/06/16/049239.full.pdf
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17:00 < fenn> it's CC-BY
17:02 < fenn> (same paper) https://www.sciencedirect.com/science/article/pii/S2329050117300359/pdfft?md5=465ce3e451288f326253fcc08400f499&pid=1-s2.0-S2329050117300359-main.pdf
17:02 < fenn> the biorxiv one is huge
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17:41 < kanzure> i wonder if there are any molecular nanotech things that would be particularly enabled by continuous DNA synthesis   instead of one-off synthesis. like feeding a continuous ticker tape to a molecular protein machine thing.
17:45 < kanzure> ribosome sort of does that already (to produce proteins), 'cept we don't have it hooked up to continuous DNA/RNA synthesis.
17:47 < kanzure> and i'm not really sure what that would be useful for.
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19:22 < kanzure> ah it has a patent of course,
19:22 < kanzure> "Shortcut procedure of transgene integration by hypotonic shock into male germinal cells for gene expression and transgenesis" https://patents.google.com/patent/US20140351966A1/en
19:25 < kanzure> .wik impalefection
19:25 < yoleaux> "Impalefection is a method of gene delivery using nanomaterials, such as carbon nanofibers, carbon nanotubes, nanowires." — https://en.wikipedia.org/wiki/Impalefection
19:26 < fenn> the stuff of nightmares
19:37 < kanzure> hypotonic loading of proteins into red blood cells ("hypotonic exchange") https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2844929/
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19:42 < kanzure> yashgaroth: let's see if this guy wants to collaborate on something http://www.niab.org.in/PeopleResearchNirmalya.aspx
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19:56 < kanzure> in this study they use "envelope-free lentivirus" on mouse zygotes and got a high transgenesis rate https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381737/    but shouldn't this work even without a viral capsid..?
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20:34 < kanzure> ha, cleared out my tabs.
20:34 < kanzure> now what?
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22:15 < kanzure> "Derivation of a human blastocyst after heterologous nuclear transfer to donated oocytes" https://www.sciencedirect.com/science/article/pii/S1472648310609625
22:20 < kanzure> "Somatic cell haploidization: An update" http://www.rbmojournal.com/article/S1472-6483(10)62056-1/pdf
22:40 < kanzure> "Reproductive semi‐cloning respecting biparental origin : A biologically unsound principle" https://academic.oup.com/humrep/article/18/3/472/626024
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22:48 < kanzure> 'chimeras intended for human gamete production through interspecies blastocyst complementation'
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--- Log closed Sun Mar 25 00:00:28 2018