Oliver Smithies:

[00:00:00] So this is the beginning of book capital H’.  May 1998.  May 28th, 1998 to Thursday, November 12th, 1998.  Continuing plasmid constructions.  Thinking about minigenes for HPRT on page nine, Tuesday, June 2nd.  Alternative HPRT minigenes.  And with Monday, [00:01:00] June 1st some overnight thoughts on future work and attached are four little pieces of paper that were written during the night.  Typically at that time I would wake up with thoughts and write them down before going back to sleep.  So here are three little sheets of paper with different things that were going through my head.  Detection of atherogenic or protective mutations, etc.  Cell type switching and renal afferent arteriolar work, etc.  So those are always to me rather precious, these waking up in the night ideas and writing them down on my old thought that during the night you re-scramble all of the information that was going on in the [00:02:00] previous day and sometimes come up with new combinations.  And these are the ideas that get written down at night.

So page 10 and 11 are really quite nice illustration of how I would think about things.  And still do occasionally.  Not so commonly.

Beginning to think about cell type switching on the 13th and adding a comment NM return day.  She must have been away.  Wednesday, June 3rd.  All-or-none cell type switching.  Several recent papers suggest that the cytoplasm may be able to switch the nuclear [00:03:00] behavior in a nucleus transfer experiment.  Some thoughts of this general type with a couple of these overnight pages attached.

Thursday, June 4th with a large cross out and counting chickens.  Evidently counting chickens before they hatch.  Unknown significance.  Think something a bit mixed up on Monday, June 8th, page 21.  Crisscross tests.  [00:04:00] The conclusion that the tubes were mixed at some step.  So they got mixed up.

And so on Thursday, June 12th, page 29 first try at an assembly which would presumably let the HPRT gene be inserted into the simian sarcoma virus locus in humans but not very well documented here.  First try at SSV [00:05:00] HPRT mini assembly.  But a speedy test and the conclusion was it was too speedy.  So here I’m going on with this repurifying the SSV9HB fragment, etc.  Trying to make this construct.  More down to earth, PCR primers for the ACE exon 10 neo sequencing Thursday, June 18th, [00:06:00] page 37.  [00:07:00]

Still working at the HPRT constructs.  Tuesday, June 23rd, my birthday, page 51.  R1 excision.  HPRT delta.  So let’s see.  June 23rd, in 1998 would be my seventy-third birthday.  Just doing ordinary experiments that a graduate student might do.  [00:08:00] Saturday, July 4th, Nobuyo is back.  Nobuyo NM return day, page 55.  HPRT promoter homologous recombination construct.  Something that in the end turned out to be quite valuable was gap repair.  Vicky Valancius did quite a bit of work on that.  On Tuesday, July 7th, page 59, there are some thoughts on gap repair modified.  July 28th.  [00:09:00] Possible use of HPRT delta by delta HPRT to assist in long-gap repair for Nobuyuki.  Presumably Nobuyuki Takahashi.  More thoughts on HPRT promoter on Saturday, July 25th , page 61.  Back from — there was a gap between July 7th and July 25th which evidently I’d been to Australia and Taiwan.  [00:10:00] The various constructs were being considered again on page 62, Sunday, July 26th.  And then haptoglobin comes back to mind Wednesday, July 29th, page 65.  Haptoglobin knockout.  Some thoughts on haptoglobin and iron transport.  Thinking it might be useful to work with the gene.  [00:11:00] Paper by Olson.  Specific expression of haptoglobin in implantation-stage rabbit uterine epithelium.  Hp and Sertoli cell, etc.  So possible idea being to make a haptoglobin transgene.  Hp 1/0 ES cell, etc., etc.  To try to improve germline transmission.  Not very well stated.

So continuing with the HPRT manipulations Friday, July 31st, page 67.  [00:12:00] The following day, page 69, Friday, July 31st, a reminder that it was my father’s birthday was July 31st.  But he wasn’t of course alive at that time.  More work on HPRT delta and other constructs being made.  [00:13:00] Thinking of using Cre-lox duplications, page 83, Friday, August 7th.  Received plasmids from Allan Bradley where he was using Cre-lox sequences.  And copies of my letters to him and of his replies.  Page 93, Saturday, August 15th.  Back to Stella models.  Masaki Ito visited for about two weeks ending August 16th.  He demonstrated that two feedback loops would [00:14:00] reproduce the renin feedback better than the old model.  So he’d been some modifications of the Stella models to try to reproduce what was being seen practically.

[00:15:00] Haptoglobin still in the mind on page 111 Friday, September 11th.  Thoughts on haptoglobin minus minus.  Randy Thresher has many derivate clones in several ES cell lines that give haptoglobin disruption.  But none go germline.  Some thoughts on what to do about it.  And beginning to make a plasmid which would [00:16:00] have the EGFP gene in the HPRT locus on page 113, September 12th.  Begin Hp EGFP HPRT delta construct.  Making the internal ribosomal entry site fragment driving the EGFP gene from starting with a commercially available plasmid on page 119.  [00:17:00] Some thoughts on September 19th, page 121 on transformation, etc., on use of adeno-associated virus, etc.  Rather complicated arguments.  It never to my knowledge led anywhere.  Continuing to make these various plasmids.  [00:18:00]

Some thoughts on Saturday, October 17th, page 145.  Is this red?


It is.


The little comment there.  Accidental color.  The whole page is written with a red pen.  And my color vision is so poor that I had thought that it was a black pen.  Though I can, looking at it now, see a little difference.  But I wouldn’t have recognized it as being obviously red.  But this is talking about cell metaplasia versus metamorphosis.  [00:19:00] Renin cell number work emphasizes the persistence or recruitment of renin-producing cell in the afferent glomerular arterioles.  That’s work done with Ariel Gomez.  And there is a little, one of these odd pieces of paper.  Whether night or not I’m not sure.  But excise the heart and dissect out the coronary with the Gomez HCL method and stain for lipid, etc.  Consider the cell type to be determined by switching on off states of a large number of [00:20:00] independent systems, etc.  Various thoughts on what might be going on.  Plasmid construction and checking continues to the end of this book, which ends on Thursday, November 12th.  Testing for contamination.  Conclusion.  A strange contaminant in all the samples.  Look for it in gel too, etc.  [00:21:00] So the conclusion 81 failed, 82 confirmed expectations.  So trying to determine what’s going on ends this book on Thursday, November 12th, page 163.  [00:21:20]