Oliver Smithies:

[00:00:00] So this is the beginning of book capital M’, July 29th, 2001 through March of 2002.  It begins with continuing work on making this construct which is called NAAC omega, which probably dates back to an earlier thought on omega transformation using an omega type construct to introduce a stem cell factor.  But anyway begins with pMC1 poly(A), etc., etc. on this page one.  [00:01:00] Different strategies for the fluorescent genes being pursued in the following pages.  Some new thoughts on Saturday, August 11th, page 15.  [00:02:00] The beta-MHC promoter driving HRGN and puromycin allows selection for fetal type.  That’s cardiomyocytes.  The alpha-MHC promoter driving CNN neo allows selection for the adult type cardiomyocytes.  And I need to have a means of selecting for fetal or adult and/or fetal and adult, etc.  Talking about different solutions to that type of problem on page 15.

Rather nice set of results almost certainly from Seigo [00:03:00] on Wednesday, August 22nd, page 19.  Various fluorescent cells.  And the ability to separate them by cell sorting so that the results with HRGNCTA and CCTA show that three filter sets, that’s one for green and one for cyan and whatever the other one was, will allow either or and discrimination.  And tested with FACS separation of ES cells that clearly separation was very easy.  [00:04:00] Must have been writing grants at that time because that’s August 22nd and the next entry is Saturday, September 1st with a relief from grant writing with a comment that later on, Tuesday, October 23rd, blue beating cardiomyocytes were observed and green.  But anyway relief from grant writing Saturday, September 1st, doing an experiment in looking at candidates for BHRGNAC.  And since I even at that time would get muddled about what the constructs are, Sunday, September 2nd, page 23 [00:05:00] is a review of the status of various assemblies which are difficult to reconstruct at this time and even then were not easy.  So various constructs being considered.

And on Tuesday, September 11th, 9/11, a comment that the World Trade Center towers were destroyed by hijacked aircraft.  It was actually something that I remember also for a different reason.  I was being interviewed [00:06:00] by a reporter as a recipient of the Lasker Award.  And the lady who was interviewing me suddenly got very distracted and said, “There’s something terrible happening in New York.”  And so the interview stopped and we all were horrified to follow what happened at the World Trade Center, Tuesday, 9/11/2001.

Returning to Stella, the modeling, Sunday, September 16th and Monday, September 17th.  [00:07:00] Looking at the old program that had the whole of the angiotensinogen, angiotensin, and ACE and so on construct.  Thinking about adding the bradykinin part because our experiments in animals more and more convinced us that ACE inhibitors for example were important because of their effects on bradykinin rather than their effects on angiotensin I and angiotensin II despite the importance of angiotensin II in controlling blood pressure.  So [00:08:00] Tuesday, September 18th on that same page a comment.  Modified the program to allow ACE inhibition as a discrete function and see what happens.  It in fact did lead to publications.  And still in the Stella mode, the following page, Friday, September 28th and later October 8th, page 37, program switching.  And this business about stable switching being considered again.  A little overnight diagram.  Almost incomprehensible.  On page 36.  [00:09:00] Talking about autophosphorylation and dephosphorylation as being the controlling element in switching.  Trying to make a program to do that.  With some corrections of the ACE inhibition equations on page 39, Wednesday, October 3rd.  [00:10:00]

Back to vectors Saturday, October 6th, page 41.  New vector, 5’ homology beta-actin HRGN poly(A) SV40 poly(A) and then to go into the HPRT locus construct.  Doesn’t look very different from preceding ones but it must have been some variant.  Beta-myosin heavy chain driving puro with puro selection.  [00:11:00] And green fluorescent protein.  Page 43 with comment that after seven to eight days in puromycin the only surviving cells are beating and they are stable.  So that it was possible to use puromycin selection to isolate just beating cardiomyocytes.  All of the cells that were surviving were beating and all of them were fluorescent.  [00:12:00]

Page 47 Friday, October 19th bingo.  First significant attempt at phosphorylation and dephosphorylation and autophosphorylation under stress worked to get a flip-flop type of construct.  But it doesn’t show hysteresis on reversal.  So it worked partly at this rather simple Stella diagram illustrated on page 46 and continuing on the [00:13:00] next few pages.  Page 49 October 22nd.  And very careful set of runs on Tuesday, Wednesday, October 23rd, 24th, page 51 which was change the scan still obtaining something right as illustrated by the sketch on page 50.  That M’53.2.9.  That was one particular version of the program was working.  So can give [00:14:00] excellent hysteresis although the meaning of it I wasn’t yet clear of.  Later on I did understand better what was happening.  So this is page 53 Thursday, October 25th attempt to alter the construct, the model, to give hysteresis.  Page 57 Tuesday, November 27th [00:15:00] I make a comment that the grant was sent off.  Probably the last ever, query exclamation mark.  But in fact it wasn’t the end of my grant writing.

At this point in the modeling I’ve not yet appreciated that the switching, the reversal was really partly an artifact of the Stella programming.  That the Stella programs would carry out different elements delta X or delta Y.  Changing delta X or delta Y or delta Z in a certain order.  [00:16:00] And that it would affect the result depending on which order the events occurred, delta changes.  I’m not sure whether this will be recorded or not.  But it’s getting closer on Friday, November 30th, page 59.  Can’t get any simpler.  [00:17:00] Page 65 Saturday, December 1st and Sunday, December 16th shows the two things that were going on in my mind at that time.  The programming.  Eight notches abrupt modification, etc.  And on Saturday, December 1st and Sunday, December 16th looking at different promoters driving the colored proteins.  The idea that I could start with ES cells and go to blue or ES cells going to green and then switch between them.  [00:18:00] Different constructs again.  Pages after pages of this same type of work.  [00:19:00] Again page 97 Friday, January 4th is typical of the complexity of these experiments.  It’s difficult to decipher from this vantage point now more than 10 years later.  [00:20:00] Nothing particularly remarkable in this part of the book.  Looking at different construct and different colonies obtained from them.  [00:21:00] With a typical again review of where things are at.  Sunday, February 3rd, page 115.  Review of the current status.  Mainly for alpha-myosin heavy chain constructs but with a little top note.  Back from flying 172s in New Zealand for three weeks.  Went with Field Morey to New Zealand and had some wild flying with New Zealand instructors who seemed to be able to — or seemed to accept risks of bad weather in the mountains that I would never have attempted by myself.  [00:22:00] I don’t think any of us from the US really would have allowed ourselves to fly visual under the conditions that the New Zealand instructors allowed.  It was a rather hair-raising set of adventures.  So that was Sunday February 3rd, review of the current status of the constructs.  One, two, three, four, five different constructs being looked at or elements.  [00:23:00] But animals are being obtained.  So on Wednesday, February 7th, page 121 is a note on two agouti female pups which were born using the M’33 construct which is beta-myosin heavy chain driving HRGN.  [00:24:00] Looking at the logic of these various experiments on page 135 Sunday, February 17th.  The logic of adult normal going to fetal hypertrophic and switch between the two.  [00:25:00] Various factors which can stimulate a normal adult cell to become hypertrophic.  Thinking about the need to find common factors that prevent all of these switches.  [00:26:00] Atrial natriuretic factor being constructs of the type being looked at on round page 151 Friday, March 1st.  ANF construct.  Thinking about insulators, which turned out to be quite important, page 153, Saturday, March 2nd at the bottom.  With Dominic it will also be good to test insulators.  What the effect of an insulator was when inserted into a construct.  And Dominic used this quite effectively in his work which was published.  [00:27:00] So ANF construct being sequenced on March 10th.  With a review of the situation on March 14th, Thursday, page 161, which is looking at the status of the atrial natriuretic factor construct which concludes this book.


[00:00:00] I’ll just look for it.  I want to see that this is a little continuing comment on page 153 of book M’ where the note is that with Dominic it would also be good to test an insulator.  And that did eventually lead to a publication with Dominic five or six years later.  Ciavatta, Dominic et al.  “A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation” in PNAS [00:01:00] volume 103 in 2006.  So that that idea did eventually lead to something.