How many times can you split an embryo?

Project 154 is essentially a massive legion of clones, and all the clones in each batch (one batch is produced each year) are identical and come from one ultimate source.

The simplest way to clone is to take a zygote and split it in half, producing two identical zygotes. (In fact, this is how identical twins happen—identical twins are natural clones.)

As I understand it, identical quadruplets can happen, so I believe it is possible to take one zygote, and keep splitting it, and splitting it, until you have as many clones as you need.

My aim is for a batch of several hundred clones to ultimately be made from one zygote—is this possible?

I'm concerned because as a zygote undergoes mitosis, the daughter cells are actually smaller, and they remain small—are identical twin zygotes actually smaller than the parent zygote? Is it possible to culture them and make the cells grow?

edited 30th Jun '11 5:58:25 PM by annebeeche

I believe there is in fact a biological upper limit to the number of identical children a single zygote can create.

Lemme look that up for you.

Edit: Search inconclusive. Although what you are effectively going for is cloning and that depending on method (even embryo splitting) has no upper limit.

edited 30th Jun '11 6:10:15 PM by MajorTom

Embryo splitting is my method, yes—what these guys are doing is they're taking an ordinary zygote and replacing its genome with a genome they wrote themselves. I'm depending on that having no upper limit, but a limit may exist.

edited 30th Jun '11 6:20:50 PM by annebeeche

yes, well specifically it's plausible enough depending on you're setting.

yes, though how well they grow is more depending on how early the split happend rather than how much was split

yes and no, to clone one would need some kind of artificial uterus or ureteral surrogate- making them grow is easier than culturing them.

oops, not sure how I missed that one.

I'm sure there's a hard number somewhere but if IVF can give you double digit kids give or take womb space, then 100 copies of one individual seems plausible.

Embryo splitting is my method, yes—what these guys are doing is they're taking an ordinary zygote and replacing its genome with a genome they wrote themselves. I'm depending on that having no upper limit, but a limit may exist.

that's a bit harder to do though, hopefully it's some time in the future.

edited 30th Jun '11 6:23:40 PM by GiantSpaceChinchilla

IVF is a completely unrelated concept.

These clones are all individually gestated in artificial uteri, collectively called a Honeycomb.

You would need some kind of uterus or gestation chamber to develop a clone and bring it to babyhood, you don't need one for the cloning process itself.

edited 30th Jun '11 6:28:59 PM by annebeeche

I remember this coming up in a discussion of the Bokanovsky clones in Brave New World.

Yeah, I remember. I'd look it up, but a friend of mine is borrowing my copy.

25%. of pregnancies with IVF are twins. (In normal population, the rate is one set of twins per 80 births.) Triplets are seen in approximately 2-3% of pregnancies. — according to this site

the numbers seem off but not by that much.

personality that's the part that concerns me. manipulating that much dna. but again, it's apparently in the future.

Still unrelated because nobody is getting fertilized, but that is an interesting fact. I wonder why that is?

The genome was written in software made for the job (called Nucleo-Type, actually) and "synth-printed", meaning that the DNA strands were chemically synthesized in an exact electronically-determined order. All the proteins that are normally attached to the DNA were also synthesized and added.

My A&P teacher has suggested that a "synth-printing" process may already exist.

edited 30th Jun '11 10:06:55 PM by annebeeche

It does, currently it's a pain in the sitting muscle that ([[[[YMMV}} seemingly]]} takes forever and you get relatively tiny strands that would take another forever to work with vectors like plasmids, viruses, or yeast artificial chromosomes. presumably human artificial chromosomes would be developed larger than 10K to 1.5 Million base pairs but even a gene gun might have problems loading it into a cell for testing verification and archiving. at approximately 3 billion base pairs that could take some time. PCR helps but not that much and you need to bash out a blue print.

Still unrelated because nobody is getting fertilized, but that is an interesting fact. I wonder why that is?

• depends on how you define fertilized, I guess.
• I think there was a European study, but for the life of me i can't remember who did it. not really hear or there since I also don't remember what they said. but it might be helpful if you want to do some further reading.

Well, apparently SC Bio has developed an efficient, failproof method for printing out whole chromosomes of base pairs. It's still a complex, difficult process, though, so they'd prefer to have to do it as few times as possible.

That's where the embryo splitting comes in handy.

Actually, I had a brainfart and mistook IVF for artificial insemination for some reason (which really is irrelevant because no one is being inseminated). Disregard what I said there.

While a test tube zygote is more prone to splitting for some reason, I'm still not sure if it's relevant because the splitting here is being done on purpose, not happening spontaneously.

edited 1st Jul '11 1:12:24 AM by annebeeche

IVF is unrelated here because it only increases the frequency of fraternal twinning. The reason is that they'd often put in multiple embryos at once because there was a low chance of any of them successfully implanting. However, when they do that, there's a chance that more than one will implant as well.

Those are actually two different things.

A zygote starts out as a single cell, then starts splitting and forming into a ball of identical cells. After enough cells have formed, genes start turning on and off in certain cell lines, causing some to become skin cells, brain cells, etc. If you cut the thing in half before cell differentiation but after it's started splitting, you'll get clones. You can do this numerous times and both balls of cells will regrow what they lost, but I think if you do it to much they'll have used up too much energy growing cells and they'll die. Not sure where that number is.

Replacing a zygote's genome is a different thing altogether. What you do is take a cell from a multi-celled pre-differentiation embryo and suck out the genes from it. You also suck out the genes from the unrelated zygote. And then you put the new genome in. You could do this pretty much indefinitely, as long as you have a steady supply of zygotes. However, clones made this way aren't completely identical, because this procedure does not duplicate the mitochondria. The zygote will still have it's original mitochondria, in a sense having three genetic parents. (I remember hearing about three cloned bulls, one of whom was considerably bigger than the other two. People suspected fraud and had them tested, but it turned out the size difference was due to the different mitochondria.)

I never said they're the same thing. That's just another thing I said they were doing. Inserting the genome first means that they have to do embryo splitting. That and I know what the processes are.

re IVF & fraternal twinning: That makes perfect sense, in fact, I know that's the reason octomom had that litter.

You're right about the mitochondria—in fact, my protagonist, who is one of the clones born from this process, attempted in an older version of the story to search for the woman who donated the egg out of curiosity since that is the closest thing he has to a biological mother, by analyzing his mDNA. The detail was since removed when I realized how impossible it would be to actually find the mother without a sampling pool of individuals.

EDIT: if you say that the reason why the cells die after too much twinning is lack of energy, then what we need is to give them more energy, right? Is it at all possible to feed them?

Incidentally enough, that was kind of what I was asking in the op.

edited 1st Jul '11 8:58:38 AM by annebeeche

[1] apparently my european study was in oxford, big oops.

also, if you can manipulate something as huge as the human genome then why not the mitochondrial genome?

just be sure to spend more than 2200 USD on you're pet project [2]

I think what the OP might want to try reading up on is telomers. A molecule at each end of each chromosome, which gets shortened each time the chromosome is duplicated (i.e. in prep for cell division). Apparently this may be a component of biological aging, that telomers become too short, but I'm sure it's not the "whole story" on aging.

As for artificially split cells being too small, I wouldn't worry about that. I'm confidently guessing that cells have some idea about how big they "want" to be, and if you take a fertilized egg that has divided once, and you split it up into two separate cells, then they'll absorb water and nutrients from the surroundings until they've grown to the size they "want" to be, before dividing again.

As for plausibility, I'd say that 128 clones would work for me, but a much bigger number like 4096 might not, although if you talk about telomers and so forth, it makes it seem as if you've done some homework, and that makes it easier to swallow.

If there is a telomer problem, it's probably going to be shortened lifespan. I'd expect the clones to mature normally, rather than with unnatural rapidity, but they might start growing old when they're in their 40s or even 30s, rather than when they're in their 60s. But I'm not 100% sure that telomer shortening begins to happen right after fertilization. I *AM* pretty sure that homozygotic twins don't have shorter lifespans than heterozygotic twins, because if that was the case, I'd have heard about it or read about it, somehow, and I haven't.

Perhaps the telomer thing is only an issue with Dolly-style adult cloning? The OP might want to look into adult-cloned animals to see what their lifespans and long-term health are like.

The cells running out of energy due to too much cell division doesn't make sense to me. I have to admit I don't know how the early egg is provided with carbs and micro- and macro nutrients. Later on there's the umbilical cord and the... I think placenta is the English word for it? That way the fetus gets whatever it needs of glucose and amino acids, various fat-type molecules, and minerals, from its mother.

But I don't know about the early egg, or rather the very early stage of pregnancy, because presumably the umbilical cord doesn't form right after the egg has divided itself once.

Still, if you've taken the egg out and put it in a petri dish or similar, you should be able to "feed" it by putting some glucose and amino acids and so forth into the dish, which the egg will then absorb through the cell membrane. So no problem htere. The energy thing is unscientific nonsense.

As for messing with mitochondrial DNA, the problem is that each cell contains many mitochondria. I don't remember how many but if you said thousands I'd believe it (it probably varies a lot from cell type to cell type). Taking each mitochondria out, as a physical action, and replacing its DNA, then putting the mitochondria in again, strikes me as silly. It makes more sense to "hack" the mitochondria with a virus. If the OP wants a cool term for such a tailored virus, he can start with the word "retrovirus", and either use it outright, or else hop over to English Wikipedia and read up on it (I'm sure there are thousands of words on vira, and probably a separate article on retrovira) and scan for terms that are suitable technobabble.

The one animal out of the homozygotic triplets being larger than the others, due to different mitochondrial DNA, makes some sense to me. Mitochondria are about making use of energy. High school (yes, this is high level high school biology, although I did have a good teacher) was over a decade ago, but IIRC it's the mitochondria that convert glucose into energized ATP molecules, which is what is used in most (probably all) of the various organelles in the cell, to have work performed.

If one triplet can do this process, utilize glucose to convert ADP into ATP, more efficiently than the other two, then it makes some sense that it'd grow bigger than the other two. I'd have thought protein intake would be a more significant factor than mitochondrial efficiency, but maybe not. If I were to take a wild guess, I'd guess that these animals (cows or something?) were fed mainly or exclusively on grass or otherwise "wilderness fed". If I try to visualize them living in a stable and getting as much grain as they can eat, to my mind's eye they won't end up very different in size. The ones with mitochondria that are relatively less efficient will simply be more hungry, and eat more of the grain.

I've heard of telomers before, but I don't know much about them.

My clones do not rapidly develop. I avert that trope like the black plague, I hate it and it's counterproductive for the proper rearing and psychological development of a human being anyway, as was discussed in the beginning of Brave New World.

An interesting coincidence! In an older incarnation of the story, I decided SC was going to write the 154s to age more quickly so that they can be more quickly replaced than regular humans. If the deterioration of telomeres speeds up aging by itself, then I guess SC won't have to force it.

My protagonist will die at the age of 29, though so he won't live to see it affect him.

Messing with mDNA is pointless anyway. If my guess is correct (I know where mitochondria and chloroplasts come from) the mDNA is for the development and maintenance of mitochondria as suborganisms.

EDIT: I'm a she.

Also, I think the number I'm aiming for is about a thousand per year. In any case, there has to be over 318 because my protagonist's ID is 154-01-0318, meaning that he is the 318th infant born in the first batch ever made.

edited 2nd Jul '11 4:30:57 PM by annebeeche

I have an idea that may help: if the clone(es) are female, you could harvest their eggs after they are all grown up, and use those to make more clones. You are replacing the genetic code within the egg anyway, so it's not like incest is a problem.

Also, you would probably want to neuter any genetic experiments anyway: you do NOT want their "wild oats" seeding aberrant D.N.A into the general populace.

They're not, sorry.

Way ahead of you—spermatocytes are edited out of the genome, and the seminiferous tubules are altered so they cannot help with the formation of sperm anyway. They are genetically and effectively sterile.

This is also a soft spot with my (deviant and fugitive) protagonist who would like to be a daddy, but cannot have kids.

Edit: You know, it's amazing how few people consider that I've made the 154's sterile, so I'm glad you mentioned that, it shows good thinking ahead on your part. There was this one guy on another site a while back who told me that I should consider the possibility of a generation of children born from 154's and the formation of a new race, because people having sex with 154's would inevitably happen.

I told him that yes, people having sex with 154's would inevitably happen... so that's why they've been made sterile.

EDIT: And this is just a terminology pet peeve, but at this point in the game they aren't "experiments" per se, that would imply that they were mucking around when they made them or they were performing a lab study. That's not actually true because for a couple decades before they even wrote the Project 154 genome the actual experiments were on smaller, faster-growing organisms like bacteria, protists, mice, and eventually rhesus monkeys, testing the various features that the project proper would have. Nope—by the time they made the humans, they knew what they were doing.

These are scientists, after all, and scientists like to be sure and precise.

EDIT: Although it is true that the first few batches (one batch yearly) of 154s are more experimental than the later ones because they weren't precisely sure how they would turn out specifically as humans, since the rearing process is complex and much of its formation is guesswork. With every new batch, they update the genome based on their observations.

edited 3rd Jul '11 8:01:16 AM by annebeeche

it's less of a molecule and more of a region of a chromosome, the end regions specifically, it has a lot of repeats since during replication the dna gets scissored and the genome needed some ablative bits. I've heard it compared to clock punk.

true, the trick is to start early before too much differentiation has set in. kind of a stem cell thing.

if they don't want to squeeze out some more efficiency, they may want to make alterations so that they can patent the genome or obscure the identity of the donner.

mucking about with dna does that

some-batch has to be the first, besides if they went through reese monkeys, pigs, et al. they have some familiarity with the human genome since large chunks of dna are shared amongst us. Also, it probably helps that the human genome is one of the most studied with a genome library and a vast list of possible things that can go wrong.

You'll be surprised at how many seemingly separate species can reproduce, and produce even reproductive offspring at that. Worst case scenario, a 154/normal hybrid would have genetic defects (especially since some stuff is added in to the 154 genome like tapeta lucida, which means new genes that a normal person would not have), but that offspring would still very likely be viable and be able to reproduce if the genetic sterilization hadn't been done, obviously.

Changing the mDNA is too hard and inconvenient—you'd have to find and change every mitochondrium in the cytoplasm, and there's a lot of them in there. They're the symbiotic descendants of aerobic bacteria, which is why they have their own DNA in the first place.

That and finding somebody using DNA fingerprinting only works if you have a reasonable pool of possiblities—in forensics it's only used in conjunction with other evidence like physical appearance, location, family etc. Without those other details it would be like looking at every license plate in the US to find one car *

Of course, that's why they worked up the test species.

edited 3rd Jul '11 9:45:38 AM by annebeeche

fun stuff

You'll be surprised at how many seemingly separate species can reproduce, and produce even reproductive offspring at that.

no I wouldn't, but that doesn't change the fact that I had missed that they were transgenic as well.

It's just 100,000 or so organelles per ova with a genome of 16,569 or so base pairs, compared to ova cell with 3 billion or so base pairs.

a maternity test is relatively easy to do and might come up in the story. besides, they may want to patent the genome.

I'm sure it can already be patented the way it is. There's several major changes including the tapeta lucida, as well as a hair color spliced in from a Northern Cardinal.

Just 100,000 organelles? You'll have to change all of those organelles, just so you know.

Again maternity and paternity tests work because of the small pool of people to choose from. The donor could be from anybody in California—the US even.

edited 3rd Jul '11 10:05:57 AM by annebeeche

Telomeres are an issue when cloning adults not early embryos. A few dozen extra cell divisions isn't much compared to the billions it takes to grow a mature organism.

There isn't really a reason to mess with the mitochondria unless the eggs you're working with have few or defective mitochondria. In which case you can do a mitochondria transfer from a healthy cell w/o removing the original mitochondria.

I'm assuming that obscuring the clone's genetic origins is less important than growing healthy clones.

I'm sure it can already be patented the way it is. There's several major changes including the tapeta lucida, as well as a hair color spliced in from a Northern Cardinal.

I was talking about the mDNA, the nuclear DNA definitely sounds like they made plenty of changes, probably has fingerprints all over it.

Just 100, 000 organelles? You'll have to change all of those organelles, just so you know.

it's a print run of a large book like a text book, compared to the library of congress that is the human genome it will be easy.

Again maternity and paternity tests work because of the small pool of people to choose from. The donor could be from anybody in California—the US even.

or the world. what with people tracking down mitochondrial and y chromosome most recent common ancestors based on genetic drift. but, based on the discussion those databased don't seem to be coming up in story and the mega corp has to do something unethical, illegal and wrong to be a mega corp I suppose.