Oliver Smithies:

[00:00:00] And this is book G’, starting on December 13th, 1997, and running through May 28th of 1998.  Continuing, of course, with what had been done immediately preceding.  So a review of the assembly of the c-kit TNAP construct, referring back to page 139 in book F’, “TNAP fragments still to make,” et cetera.  And there we go on that type of work.  The day-to-day work of assembling plasmids, which I enjoyed very much making [00:01:00] plasmids, so this was always enjoyable work, trying to work out convenient or convincing schemes to get what one wanted to have.

Working Wednesday, December 24th, Christmas Eve, page 25, “Multiple primer try on the fusion.  N=20,” that was just the number of standard [00:02:00] reactions in the reaction mix.  It made it easier to make reaction mix accurately, and this page showed, typically, that the protocol is written out rather carefully, with so many microliters of that, and so many microliters of the other, and a checkmark after each addition has been made.  A very standard procedure in all of my experimental work, that’s to list exactly what one wants to do, and then put a checkmark as you have done it, and therefore if you get interrupted, it doesn’t matter.  So Christmas Eve, Wednesday, December 24th, there are about 10 different additions and Thursday, Christmas Day, another PCR reaction with very similar things, except slightly different primers. [00:03:00]

And here on Monday, December 29th, I have a comment, “To the UK for a new year with NM,” so Nobuyo and I went to England for the new year, and were away from December 29th to Tuesday, January 6th, visiting my family.

Continuing [00:04:00] the usual things, Monday, January 12th, “Retry at digesting with the enzyme Kas,” page 45, nothing very striking.  Making these constructs.

Catching up on the PCR positive control Tuesday, January 20th, page 59.  This, typical experiments on Saturday, January 24th, sequencing tests on the targeting construct. [00:05:00] “Standard, forward, and reverse sequencing primers for Bluescript can determine the 3-5’ homology,” et cetera.  This is for the c-kit junction, for the TNAP construct.  Little do I know at this point, that I don’t think TNAP ever worked in the end, but we’ll see. (laughs) By my recollection, it never came to anything despite all the work, but that will no doubt turn up.

More sequence data, page 79, January 27th, Tuesday.  Pages and pages on TNAP, kit. [00:06:00]

Final control construct checks, Monday February 2nd, 93, page, various probables.  Next day, “Shows improvement in final step.”

Thursday, February 5th, 101 page, review of the final step. [00:07:00] So Thursday, February 12th, page 113, just one more step.  Purification and ligation for the target construct, and the review of the available fragments, various things look quite good.  Still at it on Sunday, February 15th.  Remake [00:08:00] of the fragments, TNAP c-kit XO fragment, target and control, TNAP c-kit again, good cuts.  By various tidying up jobs, the following Tuesday, page 123.

Test of a ligation on page 129 with, on the opposite side, one plasmid pattern that I thought was correct, and it’s labeled in heavy blue, “Target.” [00:09:00] So tests of it on the following page, and there’s the control.  So the targeting construct and the control.

One more sequencing check, Tuesday, February 24th, page 135.  Of course, it never is one more check, because on page 139, there are more TNAP-kit sequences.  Page 141 is quite a good page, because the construct that it had turned out to be a very useful, what I called “DupDel,” the construct that had enabled me to either duplicate a region or [00:10:00] delete a region, and we made standard plasmids to do that, and there’s been a little uncertainty because Kathleen Caron, who was a postdoc with me, had had some problems with the DupDel sequences, so four standard preparations were made on that page, or on page 140, DupDel Blue, Dam minus, DupDel Red Dam-minus, DupDel Blue Dam-plus, and DupDel Red, Dam-plus, which, Dam being the ability to survive in different strains of E. coli.

So these sequences are being confirmed [00:11:00] on page 143.

Thinking about the mouse HPRT locus again, on page 145, with R on the opposite side, reprint, or a Xerox copy of a paper published in Genomics, volume 42, in 1997, which was an inactivation of the mouse HPRT locus by a retroposon insertion and a 55 kilobase gene targeted deletion, and establishment of [00:12:00] new HPRT-deficient mouse embryonic stem cell lines.  Hirohisa Tsuda was the first author, and I’m somewhere in there, and Aya Jakobovitz is the senior author, that it was published.

On the following page, some new thoughts, Friday April 17th, page 147, a new direction for homologous stem cell gene targeting.  “Summary of experiments carried out with Koji Nikkuni shows no indication of gene targeting at the HPRT locus, and no transformation,” [00:13:00] et cetera, so what are we going to do about it?  Some various thoughts about what to do.  So designing new targeting constructs, containing the GFP gene, and targeting the HPRT locus on Friday, May 1st, page 149, first GFP, HPRT-mini design, so something that can be used to insert at that locus, and give a GFP signal at the same time.

A digression — at least not a digression, but a progression into the animal side of the work on page 151, Friday, May 1st, blood pressure measurements.  This is looking at the [00:14:00] machine that was being developed by, if I look at the machine being developed by John Rogers for us, recent data suggests a systematic decrease in blood pressures measured by the Rogers machine, versus old data, but the difference is about 10mm of mercury, so changes in the algorithm may be responsible.  And the new idea, which Jay Rogers will test.  John Rogers was the maker, and Jay Rogers, his brother, was the programmer, to try to find a way of making sure that one really [00:15:00] did measure blood pressure correctly.  That machine eventually was widely used, and was quite accurate, and reproducible, was published with, as I recollect, John Krege being the first author on describing that blood pressure machine.

And some thoughts on what is happening in relation to blood pressure on page 153, Friday May 1st, that one of the best ways of understanding what’s happening in hypertension, [00:16:00] or what’s happening for detecting hypertension or blood pressure-affecting mutations is to look at the level of renin in the plasma.  That was the thought, anyway.  And the renin can be measured on retro-orbital venous samples without anesthesia, and a list of all of the ones that we wanted to measure.  Variations at the AGT locus, angiotensin, ANP, the atrial natriuretic peptide locus, and natriuretic peptide receptor, A-locus, and the clearance receptor MPRC, ATR1a, ATR1 cell, and ENOS Cox1 and [00:17:00] Cox2, many different mutations being made and considered for study of blood pressure variation.  And hold there for a moment.

Here, on page 155, is an attempt to incorporate some features [00:18:00] that are used by the adeno associated virus AAV to insert into a specific locus, in the inverted terminal repeats, to try to get the HPRT gene into a specific place, so ITR sequences, inverted terminal repeats, and Seigo Hatada was going to make it.  So, more detail of this idea on page 156, Monday, May 25th, in 1998 [00:19:00] the first construct for homologous recombination that is length-compatible with AA virus.  I think I must have been thinking of putting the DNA — packaging the DNA into an AA virion and use it, because here is the construct, which would correct the HM1/HPRT deletion, so 5’ homology, and then the missing exons, there, one, two, and three.  So, an attempt to combine AAV and HPRT targeting. [00:20:00]

And that, continuing that work leads us to the end of the book on Thursday, May 28th, page 162.