Oliver Smithies:

[00:00:00] So here we are, I’, November 26th, 1998, going through June 11th of 1999.  I think we are going to see a continuing of various attempts at altering gene targeting frequencies, or making variants for blood pressure work.  So the book begins on Thursday, November 26th, Thanksgiving Day, working on Thanksgiving Day, page 1, new project: CRLR, calcitonin receptor-like receptor.  This is work in relation to Kathleen Caron’s work.  She had been working with adrenomedullin [00:01:00] and the involvement in that system of the CRLR receptor, and so it starts off with, “After review of the adrenergic receptor experiment with mice, I conclude that variation in these genes will affect cardiac responsiveness, but not resting blood pressure.”  No, I guess this really isn’t the — oh yes, it is related to capping.  “For example, Masert Cavale find that blood pressure and aortic responses of an adrenergic receptor-null mouse is about 55% normal,” et cetera, et cetera, “and Kathleen Caron’s choice of adrenomedullin remains good, [00:02:00] as wide variations in its level are seen under a variety of situations.  In many ways, it and the calcitonin gene-related peptide, CGRP, are like ANF, and BNF, the atrial natriuretic factor, and B natriuretic factor.  The receptor is therefore a good target, but not well-understood until last May.”  So they decided to work on that receptor gene. [00:03:00] With a note added March 13th, that’s three, four months later, enacting CRLR will also affect responses to the calcitonin gene-related peptide, which is also to some degree vasodilatory, and cardioprotective, so the combination will be interesting.  So they’re working on a different gene, CRLR, which I do remember making with some enjoyment.  So it’s beginning on November 28th, PCR for the probes, page 3.

More thoughts on the receptor, page 5, with a comment that, “the chances are that somewhere else is also knocking out the gene, so it would be useful [00:04:00] to combine it with a fluorescent, or other tag.  A modified OS — DupDel would be worthwhile, for example.”  And it shows a DupDel construct with an internal ribosomal entry, sequence-driving, enhanced GFP gene.  And so with a changing OS DupDel on page 4, 2OS-NIE OS-Neo-IRES, internal ribosomal entry site, enhanced GFP.

So, working at that construct in the following pages of CRL, R probe, Tuesday December [00:05:00] 1st, page 9, and continuing, page 11, and the different pieces needed to make the construct, following pages.

Comments on the sequences of the human calcitonin receptor gene.  The exons, many exons, at least 13 exons, and the intron length, such a complicated gene. [00:06:00]

Back to a little bit more work on TNAP-kit, for targeting.  Nobuyo is suspicious of the primers used.  She designed two more using the sequence data, and preliminary tests on NM primers, Tuesday, December 22nd, with an explanatory two or three pages attached on page 23.  But, some sequencing there, but in the wrong direction, [00:07:00] “Result confirms expectation, but rather a waste.  Need to go into the intron, not out from the intron,” Intron 2, December 21st, Monday, page 35.  So, new primers to go the right direction.

Saturday, December 26th, Boxing Day.  Used to be cigars and rugby, when I was a kid, as it comments.  TNAP, details of the TNAP sequence there, with the different primers. [00:08:00] Tests with these new primers, TNAP-kit on the real sample, page 43, Monday, December 28th.  “Controls are fine,” is the conclusion.  But nothing else is promising.  Southerns for TNAP on Monday, December 28th, and continuing in the same way.

So, Wednesday — January 13th, page 51, TNAP-kit-neo construct.  [00:09:00] Back to CRLR, Sunday, January 17th, page 53, trying to amplify known sequences from the rat CRLR exon 3 and polymerize it for a probe.  But some worry about it, that the reasoning is partly incorrect here.  The template could be either the present intron, or rat tail DNA, et cetera.  And more PCR for CRLR probes, the following page 55, purification of the fragments.

An exon 3 probe on [00:10:00] January 21st, page 59, Thursday, and some use of it to try to detect positive colonies on the following page. [00:11:00]

Page 69, Thursday February 18th, a review of homologous and non-homologous recombination experiments, with OS and Randy Thresher.  In assembling the data for publication, we carried out the left page illustrative series of analysis, which, allow fairly certain identification of the reduction in homologous versus non-homologous recombination, when exonuclease destroys the 3’ hydroxyl DNA strand and with heat shock, though I’m afraid we never did publish eventually, but we tried.  [00:12:00] And the following pages, some more of the same thing, page 71.

Back to cell switching thoughts, page 73, Thursday, February 25th, more cell switching thoughts.  In reviewing Kim’s angiotensinogen heterozygous knockouts homeostasis paper, it was pointed out that afferent arterial, the smooth muscle cells can be induced to metamorphose to renin producers by a drastically low salt diet.  In fact, the effect is small, and it’s mainly efferent arterial, et cetera, et cetera, but thinking about what [00:13:00] might be done, possibilities to induce renin-producing cells in the afferent arterial by Losartan to check their MAP-kinase phosphorylation, et cetera.  So thoughts on the animal experiment.

Back to work on Randy’s systems, attempts to rationalize the control homologous recombination to control non-homologous recombination data.  Still screening for CRLR phages, Friday, March 12th, to get out the [00:14:00] sequence from genomic sequence.  Sylvia Hiller carried out a double lift for use with a lambda library of cephitism black-6 DNA with the probes that we’ve made, and got one very good colony, or plaque, that’s to say.  And repurified, plaque pure.  Very nice return to isolating a gene from a total genome, enjoyable.  Page 77 and 76.

Modeling, began to think about combining different models of the [00:15:00] Stella type.  For example, the renin-angiotensin system, the MPR system, the Nos system, in a way, three-dimensional construct from two dimensional plains, and never did manage to carry that out, but it was trying to combine the two-dimensional Stella models, different system, or how they connect to each other by using a vertical connection.  Quite a nice idea, but never worked out.

Here we are on page 81, Thursday, April 1st, phage count rising, the CRLR phage, [00:16:00] and with detailed sequences on the rat CRLR holding sequence.  But the danger of mixing rat and humans is that rat and mouse is emphasized on page 82, which is a CRLR exon 2/PCR attempt, starting with Sylvia Hiller, has excellent lambda phage preparation, on which sequencing and PCR can be attempted.  But on the left-hand side of that, page 82, double [00:17:00] asterisks, in capitals, “BUT REMEMBER THIS IS MOUSE, NOT RAT.”

So, that sort of distinction is clear on the following page, where the heading is Saturday, April 3rd, in square parentheses, “rat DNA and mouse phage being worked on.  And a comment on Nobuyo’s birthday, page 87, Sunday April 4.  Sequence data, or lambda CRLR.  This did lead to a successful conclusion, I’m glad to say. [00:18:00]

More mouse sequencing of CRLR on Thursday, April 8th, page 91.  Four sequences, no good.  Possibly I made an error, and only good sequence was with the different point.

So, subcloning the lambda [00:19:00] adrenomedullin clones, I think that’s probably CRLR really.  But anyway, the conclusion is, none have the insert.

Page 97, things look better.  Monday, May 3rd, 99.seq with I’86 blue gave a fine sequence.  Continue.  So there is the sequence data problem, this clone. [00:20:00] Which is analyzed on the following page, ’99, Wednesday, April 28th, CRLR, exon/introns.  Inspection of new data from sequence 96 in comparison with RatC DNA, leaves little doubt that exon 2 is short and in a diferent frame from exon 3.  So that the following construct could be good.  So there’s a different frame between exon 2 and exon 3.  But exon 1 and 2 are in different frames, but exon 1 and 3 — it’s not completely clear, the consideration of the frames of the different exon, [00:21:00] as to which will be useful is there on that page.  Thinking about alternate splicing, and normal splicing, so there’s obviously something funny going on here.

So, continuing with other thoughts, and more cells, more thoughts on cell commitment, Sunday, May 9th, page 109, [00:22:00] beginning to think about cell commitment again, biphasic on/off with feedback of various sorts, and with an overnight note on page 108, “Feedback stability, absence of inducibility,” et cetera, wondering about cell commitment.

Back to the real world in a following page, more sequencing, Monday, May 10th, of one of the candidates, 5A.  “All three sequences failed. [00:23:00] Repeated, again on page 115. 103, 104, 105, 106 sequences, 107, 108, 109, et cetera, failed.”

More thoughts on the construct, page 117, Saturday, May 15th.  Continuing with the construct design again on the following page.

Back to cell commitment, more thoughts on cell commitment, Tuesday May 18th, 121 page. [00:24:00] Autocrine allows the cell to maintain its “flipped” or “flopped” state, is the thought, who knows what that means?  An attempt to extend that thought on page 121.  On flip, flop, and temporary adaptation, and long-term homeostasis, some dreams.

Some changes in the sites, blue script every now and then needed, Thursday, [00:25:00] May 20th, page 127.  A new gel extraction kit protocol being used on page 128.  Nothing very remarkable in the following few pages.  Just continuing to try to isolate the various fragments.

Some thoughts on kidney modeling, Monday May 31st, page 137.  Nobuyuki Takahashi observed no effect of heterozygous loss of the sodium potassium [00:26:00] 2- chloride co-transporter, no effect on blood pressure expected.  So that was quite a nice paper came out about in the end.  But the transporter moves to the cell membrane, and complicates for the less cytoplasmic product.  Trying to work out what might be happening on page 137.  We did publish, and it was a nice paper. [00:27:00]

Continuing isolating fragments and sequencing.  Tuesday, June 8th, page 151, assembly of various fragments, but a note, “Premature.”  Trying to do these multiple fragment assemblies, which in the end turned out to be a very [00:28:00] useful procedure.  So Thursday, June 17th, page 153, changing a site in DupDel from having two Asci site, to one site, a specific change, a nice little rest in a sense, to make what was needed, but the conclusion is, “Nothing there.  Start again.” [00:29:00]

Coming towards the end of this book, still continuing with transformations, et cetera.  And the book ends on Tuesday, June 15th, with changing a HindIII site to a ClaI site in one of the plasmids.  And so we end this book.