Oliver Smithies:

[00:00:00] Here we are on book Greek mu(μ), starting on May 1st, 1986, and running through October 28th, and with some fairly extensive changes in direction in this book.  So, still trying to ligate delta-beta and delta-beta-S on the beginning of the book, continuation of the same type of thing on page 1.

Going on here, Saturday, May 3rd, and May 5th, and so on, and so forth, different attempts at this, beta-S-K117 [00:01:00] page 11, “Check on Phil’s preparation,” nothing very remarkable at this point.

Typical comments, re-attempt at P delta-beta-S-K117 Kpn Sal, talking to Kiap, page 17, Tuesday, May 27th, continuing to check the material.  Nothing very remarkable.

On page 25, Friday, June 20th, I see something a little different, we’ll see what this is. [00:02:00] This is the beginning of looking at colonies made from – ah – I missed a page; that’s why it doesn’t make sense.  If we go to page 23, Sunday, June 15th, and this is beginning to think about what can be made from the EK cells of Martin Evans’ experiments with EK cells, what we now call “embryonic stem cells.”  So, these cells were grown on an STO cell, on STO cells. [00:03:00] These cells were plated and then radiated or treated in similar ways to prevent them from growing.  And these were — without killing them.  And these were feeder cells for the embryonic stem cell.  And so it’s starting on Friday, June the 13th, with — Natalie trypsinized, a suitable EK STO cell, allowed them to adhere, and to the supernatant, etc., two dish in which various things were plated to look at.  And so she plated onto these cells the EK STO cell without a feeder layer, and I got this, a little one.  And she plated EK STO cells, [00:04:00] dish, which is a dish where the already EK cells on a STO feeder layer.  The STO cells are dead, or at least won’t replicate, and so what we’re looking at is what happens to the EK cells when they grow without a feeder layer, just for tests of this type of thing beginning, and we’re looking to see that there are plenty of large aggregates, too big for two days; they mustn’t have been single cell, etc.  So beginning to think about this idea and looking at the same cultures over time.

And Tuesday, June 17th, a comment on my sister, Nancy’s birthday, Nancy Elizabeth, on June 17th, [00:05:00] 1986.  She would have been 56 years old, or thereabouts.

So, looking at what’s happening with little illustrations on page 22.  Four days, five days, six days, beginning to understand, to see what happened.  And so here, this is continuing on seven days on Friday, June 20th, dark material, etc., etc.

But, on Friday, June 20th, page 25, the next day, Saturday, June 28th, page 24 opposing, which is now 15 days, [00:06:00] “About one to two, to five percent of the embryonic bodies have cardiac muscle beating!”  And not necessarily large bodies or complex, but can be, 0.5mL cultures were — this is a very — moment that anybody who’s ever worked with embryonic stem cell, and has seen this happen, it’s always quite hair-raising.  Literally, your hair stands on end when you first see this, that it makes you realize that these embryonic stem cells are alive, because they’re beating, and the usual comment is oh, and it’s so common is this comment; it’s almost universal that people say, “Oh my god,” and that’s irrespective [00:07:00] of religion.  It’s a comment that’s so common when people see this, and I remember — in fact, it still makes my hair stand on end to think about it, at what you feel when you first see this, and you realize that these cells are not just culture cells, they’re almost living animals, as it were.  And in fact, they are animals in tissue culture, if one thinks about it.  I often call ES cells mice in tissue culture.

Sunday, June 29th, at 16 days, getting about over, 1mL cultures, “Most of the bodies are now dark brown and look poor.  The cells are dying.  And 0.5mL, probably more are positive, [00:08:00] more of the positive are smaller, and not brown,” etc.  Brown to figure is about 6% of positive.  So, comments on what the morphology looks like.

18 days, still looking at them, and still finding beating cells on page 27, Tuesday, July 1st.  Very sparse, but one small cluster beating, and three or four clusters not beating, and scrapped that experiment at that point.

And, starting again, Tuesday, July 1st, Natalie set up, 4mL, 2mLs, 1mL, etc., colony, cultures, change the medium twice, and [00:04:00] at four days, used to collect a reasonable set of embryos, etc.  And I think about putting them into something a bit more easy to see, so put them into a micro-dish, was the idea, 24-cell — well microdish, at times called micro; it wouldn’t be any more.  I picked 40 embryos at five days, and some embryoid bodies.  And then, move them to Niel First’s lab, Carol and Bob being in that lab, beginning to think about learning to make – to do our own work in making animals from [00:10:00] embryonic stem cells, so here is the first indication that we’re on the way to making animals, deriving mice from embryonic stem cells, not really true to say making animals, deriving animals.

And here, here is a help from Tom.  This is Tom Doetschman, I’m pretty certain; it didn’t say Tom Doetschman.  But it says, “You’ll find the embryonic fibroblast preparation method,” because we, by then — this is June 18th, 1986.  There’s a little history here.  I’d approached Martin Evans to see if we could get someone to help [00:11:00] with the work going, that was obviously being planned now to go to making, deriving mice from embryonic stem cell.  And he said, “Well, I know of a postdoc who has lots of experience with embryonic stem cells, and who’s now in Germany, and looking to return to England,” I mean, “to USA from Germany, and his name is Tom Doetschman.  If you get in touch with him, he might come to your lab.”  And I did get in touch with him, and he did say, “Yes, I’ll come.”  And, this is the beginning of any recorded contact with him, so that this is, he’s saying, he has ordered the STO cells from American-type culture collection, and they’re [00:12:00] HPRT-minus, and he says, they look pretty good.  And it goes on to a comment on how to use them, preparation of mouse embryonic fibroblasts, also being another possible way of working, to use fibroblasts, and treat them.  So, here is the beginning of a contact with Tom.  And there, as I say, also on Tuesday, July 1st, the beginning of contact with a group that knew how to manipulate embryos, Niel First’s lab, in the University of Wisconsin, Department of Meat and Animal Sciences, as I remember. [00:13:00] And so, we took these embryos to his lab, and Carol commented, thinks that these were on the large side, and even the embryos were big, so, “Try some more that are smaller,” she said, “Aim at three to four days next time,” so we’re beginning to think about going from cultured cells to real — to complete organisms.

So, Thursday, July 3rd, page 31, setting up a lot of little wells.  Transferring about 40 symmetrical embryoid bodies to microtiter agar well, etc.  And comments on what [00:14:00] they look like, and a diagram of what they look like, and what happened thereafter.  That’s Thursday, July 3rd.

Saturday, first day in eight days, first day in agar, very little change, etc.  Sunday, clearly depreciating the cells, outlying cells are dead, etc., abandon.  Perhaps too large cultures, of course, a lot of other things, one would now know the difficulty of having cells in even a small dish without the right growth factors.

Saturday, July 5th, page 33, trying human chorionic — I’m not sure what — [00:15:00] no HCHO cells — no, formaldehyde, here we go.  Let’s start again.  Saturday, July 5th, page 33.  A staining tried formaldehyde, fetal calf serum, etc., etc., fixation, and just how to stain the cells.  But, looking at embryos tested, were from 1mL of 6/27 culture, and many mitoses, so one could see that the cells were dividing, and a recipe for staining nuclei is then on page 34, for Hoechst staining. [00:16:00]

So, here we go, now beginning to look at cells, and putting them into fixatives, staining with orcein, staining them with picric acid, etc., on page 35, Monday, July 7th.  Neutral red, vital dye, and PBS, bingo, that extremely intense staining, virtually black, and try a range of concentration.  So, at least, one can stain very well with neutral red.  And, that Hoechst-33342 [00:17:00], could not see it with Rich Spritzer’s equipment.

So, beginning to trypsinize cells, and adsorbed, so Natalie sub — Tuesday July 8th, page 37, Natalie subcultured the cells, and allowed the adherent cells to attach, and then took other cells which didn’t attach, so she’s separating trypsinized EK cells, and capturing those that don’t attach to the embryonic stem cell, rather than the fibroblasts.  And, [00:18:00] the following pages are devoted to looking at these types of cell.

Wednesday, July 9th, 39 page, looking at on Thursday, some free doublets, and possibly some quadruplets, but there are clusters as well, so thinking, I’m beginning to see cell divisions.

Thursday, July 10th, staining with neutral red, mouse experiments beginning, sacrificed the second and the last of, Tuesday, July 1st, implantation experiments. We’d better look back and see what that is, no signs of anything positive, so the July [00:19:00] 1st experiment was that the cells were a reasonable set of embryos; it was talking about on four days, about 20, were put into a two-day, and three-day old — three day post-plug mouse at 3PM; in other words, at two or three days DPC, “days post coitum,” though we weren’t using that term yet, but these are two [00:20:00] and three days DPC mice, and into them, we put a reasonable set of embryos in the hope that some animals would accept these cells, and so there we are.  That’s with the sacrifice of these, of the animals to look. [00:21:00] The — sacrifice the 2nd, the last of the Tuesday July 1st implantation experiments, presumably sacrificed one earlier that I didn’t notice.  The first was on Tuesday and was also negative, so that’s why there is not much notice of it, but there’s no sign of any implantation in the Tuesday, July 1st implantation experiment.  However, we also sacrificed all five of the Wednesday, July 2nd implantations, so the second implantation had been carried out on July 2nd. [00:22:00] On the 5, Wednesday July 2nd, implantation, which was not well-recorded.  And, this says, all females — sorry, all sacrifice — do it again, that we also sacrificed all five of the Wednesday, July 2nd implantation experiment, and one female had one implantation, dark red, encapsulated, like an amniotic sac, body with some structure.  No sign of a placenta, with the time, in that time.  And no signs of a placenta, but dissected out the body, fixed in  Bouin’s [00:23:00] prior to section.  “Very promising.  Try in future for 16-cell symmetrical morula.”

And so we’ll end this section by the page 43, Thursday July 20 evening cells, left most of the day at room temperature, etc., in Niel First’s embryo medium, seems to have a lot of doublets and some quadruples, and it checks out on double staining with neutral red and Hoechst, “Get more of this medium and try again, and set up for photography.”

On the opposite page, a note from, looks like Jerry, possibly. [00:24:00] “Enclosed is the,” something something gene, “gen3 gene described in MGG and a reference,” etc.. “Good luck with it,” anyway we can find out what that is later on.  And that’s a good place to stop today.


[00:00:00] OK, this is continuing on page 43, [00:01:00] of book mu(μ), Thursday, July 10th, still looking at what’s happening in the tissue culture of embryonic stem cell cultures.  And here on Friday, July 11th, page 45, we’re beginning to set up a G418-resistant feeder cells that were going to be used to feed the embryonic stem cells, STO cells as they were called.

Not very happy on Sunday, July 13th, 47, “Abandon the previous culture,” and some staining experiments as usual to stain the cell, continuing in this way to try to get photographs of the [00:02:00] cells on page 48, Monday, July 14th.  Just writing about the cultures, not quite so satisfactory as trying to get photographic record.  So here we continue doing this type of experiment.

There is an indication, or clear documentation of the fact that we were beginning to do more and more implantation experiments with these various cells, Wednesday, July 16th, page 53.  This is talking about mu-53, which is a page number, EK+STO, [00:03:00] that means EK cell on the feeder layer, STO cell, over to Laura in the lab, Meat and Animal Science, and selected, where Laura selected average-size cells, the larger are presumably STO cells, and put at eight-to-ten per blastula, into 16 blastula.  So this is 16 transfers, by error when they were returned into the mouse, there was a note that, “All 16 went into one side of the uterus; intended eight per side,” but it was neatly done. [00:04:00]

Unfortunately, on page 52, it records that “No implantation, as expected from the error.”  It wouldn’t matter in the pig, but it matters in the mouse.

So, back to feeder layers, following pages.  And embryoid body transferred from July 15th, seven days.  More work on G418-resistant embryonic cells, on Wednesday July 23rd, page 57.  So, continuing over the next many pages to work on this problem.

STO cells again, page 61. [00:05:00] We harvested some EK cells from the STO feeder layers on Thursday, July 31st, page 63, and set them up in various ways, and sent some to Laura, in Niel First’s lab, for microinjection.  So here is the comment that, “They were microinjected into about 20 blastocysts with as best as could be determined with good cells.  Not a very good day,” is the comment.  “Try again on Monday.”  This was Thursday.  “Two animals left three, right seven, left five, right five.”  But three animals were [00:06:00] born, nonetheless on 7/17, and five animals on 7/18, and one of them is a chimera, judged by coat color.  So that, transfer were, in fact, beginning to work.  Quite a technical achievement in the beginning of our being able to make mice from EK cells, or embryonic stem cells.

Another set of transfers on Monday, August 4th, page 65.  “Exciting times.”  Laura is spelled suddenly a different way, “Lora” instead of “Laura,” and I decided [00:07:00] to use cells that are settled briefly, and pick uniform spherical ones again, not easy to decide which are correct.  Nonetheless, there were two experiments done, nine blastocysts, four left, five right.  As a control, it sounds like STO cell, and then, it’s not very clear, but there is, the result is, nonetheless that two mice were born on 08/22 with no comment on whether they were chimeras or not.  And mu-55 is a different lot of EK cells.   [00:08:00] Mu-55, let me just look back at page 55, see if there’s any record of those cells.  No, these are mu-55 fibroblasts; that’s the feeder layer, STO cells, mu-55 feeder layer.  So this is an experiment with those feeder layers on Tuesday, August 5th, with EK cells grown on that feeder layer.

So, due August 17th or 18th, and here on page 66, it says [00:09:00] 8-23, “Provisionally both are chimera,” so two mice are born that appear to be chimeras from that experiment.  Getting better.

Here is a different topic again, looking for cells making hemoglobin, and particularly hemoglobin-S, Friday August 8th, page 69, detection of hemoglobin-S, sickle cell hemoglobin-containing cells among the majority of hemoglobin-A-containing cells, with a view to trying to have an ability to find the changing hemoglobin-A to hemoglobin-S in this case, because we had cultures [00:10:00] expressing hemoglobin-A, and a long time ago, used in the early targeting, and looking for the ability to get the hemoglobin gene as if we would in 2015 call “edited” to be hemoglobin-S instead of hemoglobin-A.  And the aim is to use monoclonal antibody HbS that is specific versus hemoglobin A.

And the preliminary tests will be to detect human hemoglobin A-containing cells from among mouse hemoglobin-containing cells, etc., etc., so thinking about doing that type of experiment, [00:11:00] Friday August 8th, page 71, fluorescent anti-human hemoglobin tests being carried out, and note of receiving a fluorescein-conjugated IgG fraction of rabbit-anti-human hemoglobin.  And we’ll see it in the following day, Sunday, August 10th, page 73.  And thereafter so, also some use of antibodies looking at whether an Ouchterlony plate could show antibody-antigen reaction.

A note that, on Wednesday, August 13th, page 77, “George Stam”, that’s Stamatoyannopoulos, everybody called him “Stam” because of the long Greek surname, [00:12:00] and he uses mouse erythroleukemia cells, and so on, etc.  Trying to get the technique to work at a cellular level.

Trying, using something that will lyse the red cells, lysolecithin, page 81, Thursday, August 14th, all with the same general aim.  Some more antibodies obtained from Cooper Biomedical on page 83.  And still more [00:13:00] antibodies on page 87, Friday, August 15th.

Looking at the culturing of EK cells again, Monday, August 18th, page 91, mu-to-mu series with defrosted, zero, first, and second passage cells.  And comments on the use of feeder layers.

Trying to get the ASF-21, that’s [00:14:00] cell line with the human chromosome 11 in it, and therefore, the beta-globin locus in it, page 95, Tuesday, August 19th, Phil, that’s Phil Howell, and induce some of those cells with HMBA 5mM to see if we could see the induction of hemoglobin synthesis.  “Excellent fluorescence is the comment, but no discrimination,” on page 94.

Going on with the same type of test on page 99.  But no significant improvement in discrimination, “Can’t [00:15:00] tell mouse from human or human from mouse.”

Although the next page is a little bit more encouraging, page 101 with, “Promising, that, now a fairly good identification of two populations,” and one where the mouse antibody is greater than the human in one set of cells, and the other that the human antibody gives a stronger signal than the mouse.  But not really terribly good, no images.

Still worrying about this, and having the right sort of antibody, so there’s, on page 104, there’s a reprint of a paper from Ronald Jensen, [00:16:00] Ron Jensen about monoclonal antibody, specific sickle cell hemoglobins, with monoclonals, of course, being preferable, but only just coming into general use.  The usual way being instead affinity-purified antibody, as listed with a printout from Cappel Laboratories, on page 106, affinity-purified antibody.

The following pages, more work with this type of material.  Some images again, last of these Ouchterlonys on Saturday, August 23rd, [00:17:00] page 113, with the various antibodies.  And a comment that, “They’re very weak antisera, and the cross-reaction is too great with mouse hemoglobin,” so not a very happy result.

Back to EK cell cultures, Monday, August 25th, page 115, “Testing the cells, EK cells, or transferring EK cell on the mu-55 feeder layer,” etc., and the cells are about ready to use.  And in capital letters, “The three-day passages [00:18:00] are better than longer.”

More antibody tests the following day.  “They are too small,” etc., etc.  Not much antibody left.

Continuing antibody cell, here antibodies against the hemoglobins and then EK cells, two things going on at the same time.  So here we are on Thursday, August 28th, page 123, two-times and one-time passage EK cell on mu-55 feeder layers, being talked about.  And into two mice. [00:19:00]

On the left-hand side, page 122, it talks about one mouse, mu-55 DEF, O and another mu-122, half-x mu-55.6, not clear what these exactly are.  But not very important, although one of the mice died in, with under the anesthesia.  Note, “I may have lost this round.”  Talks about the implantation.

Still struggling with the antibodies, the next page or so, two, three pages. [00:20:00] On Sunday August 31st, page 131.

Back to ES cells on Tuesday, September 16th, page 135, “Attempt at making ES cells.”  Now here is the first time I remember seeing them called “ES cells.”  I don’t know whether that’s accidental or not, or whether we were all beginning to call them “ES cells” instead of “EK cells.”  Now this really is an attempt to make the embryonic stem cells, so 12 morulae were obtained, and one blastocyst from a C3H/HeJ mouse, and flushed out by Laura and transferred to a 96-well plate trying to make our own ES cells. [00:21:00] And then, little sketches about what was happening to them.  And M1-M12, 12 different little cultures, and sketches.  This was in a 24-well plate, and Natalie was looking after it.

Wednesday, September 17th, reviewing the past EK zapping with PBS-117. Phil and Brad zapped EK to about 70% kill in the presence of linearized plasmid expressing beta-S, [00:22:00] pp-beta-S-117.  So, trying to still do gene targeting.  But in this case, with EK cells, not the fibroblasts that had been used in our earlier work.

On the left-hand side of this page, quite different comment, just the outcome of some previous experiments.  “Seven, and six mice born to the beta-SG4118R.”  No, it isn’t a different experiment, similar type of thing.  “Seven and six mice born to the beta-SG418-resistant recipient,” [00:23:00] etc. “Several are chimeras, some extensive.  ES cells again, G418-resistant-beta-S cells, they look very nice.  There were, 20 blastocysts were used, five left, five right, five left, five right to two animals, but no chimeras out of the total of eight animals born.  And just some checks of the DNA autoradiography [00:24:00] on the left-hand side.

Here is something from, I presume, Tom Doetschman, from — no, it’s not.  It’s University of Alberta, must be, Tom Wegman, three vials of purified lyophilized alpha-anti-hemoglobin-S.  Monoclonal antibodies. [00:25:00] Again, antibody work, trying to get the good distinction of red cells that are expressing hemoglobin-A versus hemoglobin-S on page 145, October 23rd, “rewashed A, my own red cells, and S-sickle homozygous cells, to see if one could tell one from another, using a rather complex assay with biotin-labeled material, and avidin, but [00:26:00] nothing useful.

Continuing with the same type of problem on page 147, a review of what we have in the way of different plasmids on page 148, a series of plasmids available October 25th, 1986, in a joint discussion with OS, NM, Nobuyo Maeda, Ron Gregg, Phil Howells and BP, remember for the moment who BP is. [00:27:00] Oh it might be, yeah, probably is Brad Popovich, yeah.

Going back to Phil’s experiments, Friday, October 24th, page 151, has induced cell, etc.  And photographed on Saturday, October 25th and the results on the next page with some photographs at last.  “And very good induction,” is commented, although looking at it at this point, it doesn’t look terribly good.  However, at the time it was pleasing.

And with the continuing on Sunday, October 26th, page 154 and 155, the results of various inductions, and more photographs on the following [00:28:00] page with 100 times, 100x view of real positive cell, that must have been the control, with clear fluorescence of some of the red cell, and with good induction on one of the cultures, not terribly good at this point, but I was pleased when I did it, summarizing the results on page 159.

As we come to the end of this book with a series of images of cells, where there’s clearly, there clearly are positive cells. [00:29:00]

And the last two pages are rough tests on 24 pooled single colonies.  Phil has several 12-colony pools, etc., and looked to see if we could see induction and the results show very underlined low-level of non-typical induction, so not a very pleasing end to this book, but continuing to try.  So that’s the end of book mu.