Oliver Smithies:[00:00:00] So this is now, we’re getting to Book B’, starting January 16th, 1994, and going through to November of the same year, starting off with a continuation of the thoughts on duplicating the atrial natriuretic peptide receptor A on Sunday, January 16th, constructs for duplication and deletion again, making the same pieces, using the same pieces and assembling them in slightly different ways to give a duplication construct, and a knockout construct. [00:01:00] That is, it’s only, in essence, a technical trick to view it and not have to make too many pieces too many times, so using the same pieces of DNA. So it starts off by checking 5′ and 3′ flanking regions for Xho sites, Sunday January 16th, page 3. [00:02:00]
So, this is just little bits and pieces of making, for example, page 13, Wednesday January 19, subcloning Sal Eag fragment, from the 5′ flanking region. Both are fine; two candidates, both are fine. One that was slightly better than the other in concentration.
Changing sites, the following page. So, page 19 here, [00:03:00] Sunday, January 23rd, on the atrial natriuretic peptide receptor A duplication all but one of the fragments are now available, but with some ambiguity in the 3′ flanking, so assemble them, etc. Subcloning pieces.
First attempts at making a complete construct for the natriuretic peptide receptor 1, changing the name from atrial natriuretic peptide receptor A, now to natriuretic peptide receptor A, so NPR, instead of ANPRA-NPR1, natriuretic peptide receptor 1, a new name. [00:04:00] First attempts at making the complete construct on page 25, Friday, January 28th. This was the ligations, blue ligation and a red ligation. And there were quite good proportions, etc.
So, first cloning of the NPR1 duplication, via cell colonies, page 29, February 2nd, Wednesday. [00:05:00] No, these are the construct, not ES cells, just still at the construct stage. A design error detected.
(laughter) On page 31, Thursday, February 3rd, B’, that’s B’24, squared minis, means repeat of the experiment previously done on page 24, minis, and with a little comment, conclusion, “Three conclusions: glorious yield of the 3′ fragment construct, probably closer to 7.1kb than 9,” [00:06:00] but here is the diagram of what was being assembled, and many, here were 54 or more colonies being looked at. I’d say, in fact, it looks like 80 colonies were looked at altogether.
But need to correct the design of NPR1 duplication, and there’s a new design on page 33, Thursday, February 3rd. [00:07:00]
February 11th, Friday, a note, page 45, “An ice storm day. But the work goes on.” Looking at preparing not H1, not HHO — Xpa fragment of the TK gene. And conclusion, “2S is terrific.”
So we go on a second attempt at making the construct for the natriuretic peptide receptor 1 [00:08:00] duplication on Friday, February 11th, page 47, second attempt at making the construct. And testing these products on the following page. “Still no good.” The conclusion, one very odd, “Whatever it is, it has an Xho1 site, an Xpa site, and several HindIII, but still no good. Let’s try the ligation again.”
Third new ligation on the following page, Sunday, February 13th. [00:09:00] “Some ligation products visible. Proceed to prepare more.”
So here we are on page 55, Tuesday February 15th, “Third attempt at making the construct. The proportions of the fragments in the construct were good and can see the ligated products. Go ahead and repeat the transformation. And see B’64 for the product.”
So retransformation, page 57, [00:10:00] and testing on page 59, and on page 61, rescue of the last available B’51 colonies, at least blue looks safe, and so testing NPR1 duplication, blue tests on page 63. One looks promising. But the colonies [00:11:00] were small on the bacterial plates.
So, repeat of the ligation, of the red ligation, one for the knockout, if I remember, on page 65, Saturday, February 19th, but the conclusion is that none are correct. Although, in doing so, another gel was run, and it looks as if B’53, the third attempt, #31 is good, see [00:12:00] later, page 69.
On page 69, we look at the NPR1 duplication, red B’55, there’s details and on the left side, page 68, “self-evident.” Conclusion, “Evident to me at this point,” so two candidates obtained out of all this. Tested on Friday, February 25th, across of red-31 [00:13:00] and blue-30, with a map of what one might expect on page 70, red-31 with all the sites shown, and that blue-20. So one for duplication, and one for knockout. And decided to re-label the B’62 blue-30, call it “blue-30,” and the red one call “red-31.” [00:14:00]
Working on blue-30 on page 73, Saturday, February 26th, converting the duplication construct to the deletion construct, etc. Some thoughts on what to do on that experiment. But worrying about B’62 blue-330 as, HO as expected, but too many Eag sites. [00:15:00] So, things are not quite as they should be.
Page 74 has a summary of the ACE situations of these experiments. I think, prepared by John Krege, very neatly typed, on what had been happening on the various days. Positive control, etc., ACE duplication with a neo reverse, etc., on different pages. [00:16:00] And suggesting thaw 3D for the chromosomes, and thaw 5A and 6D for Southerns, etc. So details of what’s to be done yet for the ACE situation.
So, looking at the renin-angiotensin system, on Tuesday, March 29th, page 77, and this is, I think with respect to the ACE gene, but it’s not very clear. [00:17:00] That, thinking about situations where 1, 2, 3, and 4-gene copy animals are normotensive, and that are, assuming that the corrections of either renin concentration changes. I began to think about some sort of product of up-and-down, so several assumptions there. And let’s go over them again, the idea being that, if 1, 2, 3 and 4 are normotensive, and the assumption too, that the corrections of either renin concentration, so that if the renin concentration changes, it can compensate for [00:18:00] the increase or decrease of another gene product. And some thoughts about how this might work.
I remember this thought being, continuing as it does on the next page, Saturday, April 1st, page 79, further numbers, assume production is linear with gene dosage, but that the 1, 2, and 3-copy animals are normotensive, and therefore, make the same amounts of [00:19:00] angiotensin-1, etc. And some attempts to integrate these ups and downs into a scheme. The scheme being written down, or actual measurements being written down in a graph on page 78, that as the gene copy number goes up, the amount of angiotensinogen goes up, 0, 1, and 2-copy with solid circles, increasing, 2-0 not being [00:20:00] quite the same as 1-1. But the renin concentration, in being higher than normal in the 1-0, and below normal, or going down in renin, is more difficult than going up, so increasing renin is easy to compensate for something low. Decreasing renin when something is high doesn’t work so well. It became later on something that we were able to test at the level of renin production by the kidney. [00:21:00]
Some thoughts on page 81 of the PCR conditions. So my test, with or without an editing Taq polymerase, with a note that on the following page 83, that the Pfu definitely can be [00:22:00] an official, Pfu being the proofreading DNA polymerase. So testing more of this Pfu on the following pages. More or less continuously.
Page 89, Friday, April 29th, PCR continued. And here, looking now again, [00:23:00] at the tail-cuff data for angiotensinogen, and gene copy number, the heart rate, and the blood pressure, very crude plots, but nonetheless, experiment #3. The blood pressure does increase from one copy to two copies, and from two to three, the effect is considerably slower, although interestingly, the heart rate decreases very much more. [00:24:00]
Kim, on page 93, Thursday, June 2nd, talking about the genotyping 2-1 versus 2-2, similar, somewhat difficult to test them. [00:25:00] Looking for some markers that might help. For example, Sylvia and Madeline tested quantitative cell for genotyping 2-1 versus 2-2. I remember we had a lot of trouble with that, genotyping, very unsatisfactory. And better, one that was to do some mating tests. For example, the better logic is that, in the F2 animals, the heterozygotes are always B6 and 129-derived, whereas the homozygotes are all B6 if they’re normal, [00:26:00] or 129 if they’re doubly-mutant. So any marker closely linked to AGT can be used for genotyping, and the idea was to test D8 mit-13, and mit-14 done by Kim looked very promising to see where things were. And that test was continued on the next page or two.
So, page 98 and 99 of data on the mean arterial pressures measured intra-arterially, and the mean arterial pressures varied linearly with the copy number of AGT. [00:27:00] Showing the tail cuff measurements were different, and that’s what I noted in looking at the paper that was later published, that the mean arterial pressure was linear, but the tail cuff pressure was a little bit peculiar.
Going back to Nobuyo’s work now on atherosclerotic plaques, Monday June 20th, page 101. It was better to use a log distribution of plaque sizes, etc. And [00:28:00] some comments on her work.
Some thoughts from Nobuyo, Monday, June 24th, page 103, on what happens in different places on the Michaelis-Menten curve. [00:29:00] And with some thoughts on where you are on the Km curve with respect to angiotensinogen, but a big note on the bottom of that page, “shingles.” I remember shingles and having to stay home for awhile, and I think it was at the time of the trial of, yes of O.J. Simpson, right, and following all, it was broadcast continuously on TV, and since I had to stay home with shingles, I remember watching the whole thing. And the debacle of the gloves, [00:30:00] which were foolishly introduced as evidence. Well they were evidence, but the idea that you could take a leather glove that had been wet, and then allowed to shrink, it got him off. But I don’t remember whether it was the trial, or the initial work with the detective. I remember it was a very fascinating spectacle.
Some more equations on page 105, trying to think about how the system might be tied together. [00:31:00] Beginning to think about clearance more, page 107, so here, on Tuesday, August 2nd, thinking about the atrial natriuretic peptide clearance receptor. This was the receptor which had taken the extracellular domain, would bind atrial natriuretic peptide, but there was no function or intracellular portion, so people called it the “clearance receptor,” beginning to think about it would be useful to work with. [00:32:00] So, got some primers that might be useful for isolating that gene.
And here we go on again on page 109, Wednesday, April 3rd, 3′ end of the ANP clearance receptor, sought using different primers. Summarized on page 108 and on the following two pages in more detail.
Probes being made on Thursday, August 8th, page 113 and 115. [00:33:00] 115, typical conclusion, “Something wrong, looking back,” etc. “But the gel isn’t good,” etc., “Re-run.” Repeat of that, attempts on 7L-8R clearance receptor, page 117. Still not happy with the gel.
Prep gel, Monday August 8th, 119, nothing very special.
But a gap to the next entry being Monday, August 22nd, with, “Back from [00:34:00] the West Coast adventure.” That was Monday, August 22nd, page 121. “Two hypertensive papers off to the journal, and back from this West Coast adventure with Field Morey and George Bandow. Shingles are now 95% finished.” So here was the receptor probe, look at the 5′ clearance receptor probe.
Now, beginning to think about what happens if a promoter is duplicated. Since, the angiotensinogen [00:35:00] locus in humans turned out to be different in the two human variants, the methionine-threonine variants, were linked, tightly linked to changes in the promoter. So, as page 125 talks about the promoter duplications, over the last few weeks have been considering how to compensate for a decrease in beta-globin expression consequent to selective and/or advantage genes within the locus. One possibility is to duplicate the whole gene. “Can’t make a useful sickle-cell model this way, or [00:36:00] to put in a locus control region, or to duplicate the promoter,” and the thought of trying to alter the level of gene by duplicating the promoter. We thought, “Let’s try this with two promoters.” The easiest way is to test it with the neo gene, or test it in the HPRT locus, so can have a neo gene with one promoter, or a neo gene with two promoters, all in the HPRT locus, using that as an easy way to get a single copy of the neo gene. So go to using 14 cells, HPRT-minus, [00:37:00] make them HPRT-plus, with a neo gene in the insert, or with a neo gene driven with one promoter, or a neo gene driven with two promoters.
So, I went back to Thomas & Capecchi’s paper in Cell, where they describe detail of their PMC 1-neo promoter, which is actually complex; it’s not a simple promoter. It contains the polyoma enhancer, TK promoter, driving the neo gene, but it is possible to understand what it is, and page 126 shows the cassette two enhancers, and the SV-TK promoter, with the sequences. So I can use that [00:38:00] for the tests. So here, I’m trying to make two copies of the promoter driving the neo gene on page 129, which I called neo/neo and pro/pro, promoter/promoter neo, duplicating the gene, or duplicating the promoter in front of what, so two copies of the neo gene, or one copy of the neo gene driven by two copies of the promoter was a thought. It’s continuing to make this on page 133. And, the following pages. [00:39:00]
With possible products being considered on Tuesday, September 6th, page 141, diagnosing the construct, KH21 neo was the standard construct, with enhancer-promoter, etc.
So, we continue on page 143, trying to get the neo-neo repeat. [00:40:00] An assay made on page 142, transforming, looking for a change on the occurrence of G418 and resistance, in other words, targeted. This assay, dissectional differences.
Papers [00:41:00] and grants finished, page 145, Tuesday, November 15th. “Grants and papers finished.” And back to work on the ATR1a duplication, looking at Masaki and Tom, Tom’s success with the ATR, that would be Tom Coffman, the ATR1a disruption and its effect on blood pressure. And a look at where things are.
Initial plans for construct on Wednesday, November 16th, page 147. [00:42:00] The homology of about 8 kilobases. Not everything is labeled clearly.
Making changes on the genes, PMC1-neo, altering sites, Friday, November 18th, page 155. [00:43:00] And 159, the book is ending, by looking at the orientation of #3 and #12 from B’159, Thanksgiving Day, stained and photograph, with the final map, #3, B’159, #12, B’159 showing a map of these products. [00:44:00] [00:45:00]
So, I don’t remember exactly where I was, but [00:46:00] here ending, page 163, Wednesday, November 23rd, 1994, with these various constructs that contain a part of the ATR angiotensin receptor 1a, or the NPRa 1a receptor. They differ in their orientation. The blue-prime has an Xpa fragment, and the red one is a Bam-Bam fragment. Their orientations are the same; the ends are different. One is an Xpa/Xpa fragment, and the other is an Xpa/BamH1 fragment [00:47:00] in the vector. So Xpa/Bam, and Bam/Xpa really are the functional parts in these two clones, and that ends Book B’.