Oliver Smithies:[00:00:00] This is capital D’ book, starts July 5th, 1995, and runs through Christmas Day of the same year, 1995. Beginning with a continuation of isolating the lamB gene from Shigella, as red 6 D’3, PGKSS-lamB. And the E. coli one, which is a C’159 blue-4, and the Shigella one is D’3 red-6, [00:01:00] as listed on Wednesday, July 12th, page five. So two clones, a red one, and a blue one. The red one being Shigella, and the blue one E. coli. And they’re OK to use.
Starting a P dupdel, the duplication-deletion plasmid which can be used in two ways. Provisional scheme is listed on page 6, Tuesday July 18th. And it begins, goes on page 9, “Started dup del.”[00:02:00] Wednesday, July 19th, page 11, ATR1b, angiotensin receptor 1b again, with a note “Yipee 3!” The Southern looked good, and PCR looked good to the conclusion, “Looks very,” two underlines, “good, for 10D. 10D looks very good.” More PCRs on that clone, 10D and 10E. 10D is still positive, and 10E is also positive. [00:03:00]
“Denise will thaw out 10D and 10E and inject if the chromosomes are good.” So these are two colonies, ES cell colonies. Ligations and transformations for SB64/PGK-TK transformation using DH5-alpha cells, D’3. [00:04:00]
Twenty-four tests, stoichiometry looks good. But, the conclusion is, “Try another twenty-four.” So a restart of SB/TK on the following page. And PCR for ATR1b a couple of pages later, Sunday, July 23rd, page 19.
Yipee again, 4C is positive. Not so good, though. [00:05:00] “May repeat later; meanwhile, proceed.” And so next page is more PCRs. “Agrees with number two, but now convincing,” some more candidates about another twenty-four. Various ones were tested further by Sylvia, but all were negative. [00:06:00]
Dup-del continued on, remembering my mother’s birthday on August 9th, Wednesday, page 27. What the final product of dup-del would be, and that’s a good map of it. Various stocks, made of D’26 blue, and D’26 red. These are primers. [00:07:00] No, that’s not correct. D’26 blue, and D’26 red are not primers. They are candidates of, delta-SB64/TK and reverse.
Some insertions of sequences into the various places of loxP sites. Linker-1 insertion on page 31, and 33. [00:08:00]
Now, on Thursday, August 17th, page 37, I’m talking about angiotensin receptor 1a. [00:09:00] Using the right sort of primers, specific for ATR1a on page 36, there are five differences in the blue primer, and eight differences in the red primer, between ATR1a and ATR1b, so this is ATR1a.
Now, back to ATR1b Southern on page 41 [00:10:00] Friday, August 18th, a nice Southern, it was fine, 4a is probably a positive, with a mark on, not a perfect Southern, but is probably OK.
Looking at the orientation of the linkers being put in on page 45, and then on page 47, insertion of linker two into the plasmid, [00:11:00] with two blue ones, D’46, 33 blue and 35 blue as backup, and D’46, 22-red, conclusion that, after inserting the linker, it looks fine, and just like last time, except that I now can see the oligos.
Checks on the orientation, page 49, and first try at completing OS-dup del on August 29th, Tuesday, page 51. On the left, a big notice, “Abandon,” got distracted.” [00:12:00] Second attempt at a neo into dsp64/TK, L1 L2 to put in neo between the L1 and L2, conclusion, “It looks good,” delta-sp, conk and preparing the fragment.
And second attempt at putting the neo into OS-dup del, page 55. [00:13:00] “Just a trace of dimer visible, can’t see anything else. Try again with cold precipitate if necessay.” “Conclusion: struck out. Try another 24.”
So, Friday, September 8th, third gel screened more, got four positives. 5 and 16, correct orientation, and 19 and 20 are the reverse orientation. So, this is called, “dup-del 5.” [00:14:00]
Repeat of the ligation on page 57 for dup-del final, “Very slightly encouraging.” So here we have the construct OS-dup-del, which was 6-1 42 base pairs. Always dup-del was an important plasmid, which was used then for duplicating and deleting various things. Let’s stop for a moment.
So OS-dup-del was used a long time, and could be used for either making [00:15:00] a duplication or a deletion, with positive and negative selections, so —
And many others. And, we used as a backbone.
Yeah. It had a PGK-driven TK gene.
No, not the PGK.
Yes, it did, Nobuyo.
TK was weak. Or TK was strong and neo was weak.
Just look here. Here’s the map.
FPMC 1 — yes.
So it was dup-del 6142, PGK/TK. And the ampicillin resistance gene, and MC1-neo, so it was used for positive/negative. [00:16:00] And there were various forms of this used, slightly different form. This was dup-del-5, D’55 red.
So now, back to thinking about ACE, Friday, October 6th, “Cox1 and Cox2 papers accepted by Cell.” That was an interesting business, because those two papers were written by — one for inactivating Cox1 gene, and the other for inactivating Cox2, one with Bob Langenbach, and the other with Scott Morham. And I talked [00:17:00] to Ed Lewin at Cell about the two papers, and he wanted it as one paper, with — and I said that wasn’t fair to the authors. They’d each done separate, he says, “I don’t care about fairness, you should combine them.” I said, “OK, in that case, I’ll take the papers somewhere else.” And he yielded, and accepted the two papers. So that was a rather nice occasion of oneupmanship in getting the papers published. But we were in a strong position. The genes were of interest to many people, and we had knocked out the two independently. And so, that is what the origin of that particular comment. [00:18:00]
Now testing various ACE plasmids, beginning on page 63, John Krege’s clones. More diagnostics, [00:19:00] page 67, and diagnostic PCRs on page 69, Wednesday, October the 11th. Six oligos were designed, and made to test the exon-12, exon-12 testis, 12T of his, and exon-13 to make sure that the ACE was what we wanted. This was successful, the result being there that all [00:20:00] worked except number one. April 3 gave 274 base pairs, and E plus F, those are the different primers, E plus F gave 191 base pairs, and those were — and they were tested on eight colonies. All but one was — were correct.
Starting on the 5’ fragment, and the October 15th, page 71. There’s tests on multiple on [00:21:00] Monday October 16th, page 73. And Denise tested red-6 Shigella lamB transformed cells, and normal BK4 cells. And, with a note on Wednesday, October 25th, that Hyung-Suk Kim tells me that not surprisingly, there’s considerable variation in the level of expression at the RNA level of [00:22:00] maltoporin. For example, the blue-E. coli cell line, 3D is a 1+, and the red-S Shigella cell line, 2b is 3+. Most of the others are negative, but some are plus/minus.
So Friday, October 20th, page 77, discussion of the preparative digests and plan, looking for exons 13 and 14, of the ACE gene, 5’ fragment being Bam BLP [00:23:00] fragment of 3.2kb for exon 12, and a Bsa/Bam fragment 3 kilobases for exons 13 and 14, from the ACE gene.
Working on the 5’ fragments, Saturday, October 21st, page 79, going on with 5’ fragment. Next page, 81. And, HpaI digests of OS dup-del on [00:24:00] October 23rd, Monday, page 83, transforming, the transformation D’82, 24 minis. Only #20 is a candidate. And it looks more like a dimer. Set up another 24.
Beginning to have some crazy ideas about making renin-angiotensin system diagrams on Tuesday, October 24th, page 85. “Should be able to set up equations specifying the effect of template number on the concentration of Agt,” et cetera, et cetera, “incorporating Michaelis constants and sketch its plots,” et cetera. [00:25:00] So my idea to see what was happening. And what they would happen to, how many copies of the gene there might be, one, two, three, four, what happened to the AGT concentration, and what renin might do, and what ACE might do, what the receptor might do. And so you get to the end, you have a sort of diagram of the different things that happened when the gene dosages change, not a linear relationship, because you have to go from the gene dosage to the expression, and then you have to go the effect the renin, and you have to deal with the effects of ACE, and then with the [00:26:00] receptor, because trying to think about a diagram, diagrammatic way of dealing with the situation.
Second transformation of dup-del on page 89, “Start again.” Page, following page, “No signs of success, better go back,” et cetera. Sunday, October 29th, new digests of dup-del in the base, “All cuts are good, proceed.” [00:27:00]
So, October 30th, Monday, page 95, a prep gel for the 5’ fragment and a dup-del vector.
Thursday, November 2nd, page 101, new approaches to getting what I will call 5, “Made a linker that would replace this,” et cetera, et cetera. Silly. “Still need a straight BLP,” et cetera. Various cancellations, not happy with that idea.
A new start on the 5’ sequence into dup-del, [00:28:00] page 105, Friday, November 3rd. Continued on the following page, 107. Many little notes. A problem in remembering what sequences are controlled, control maybe some restriction enzymes, conceptual design problem on the linker on D’100, et cetera. PLPC pointers, the two sites have different sequences, [00:29:00] the 5’, the 3’ sites. So two asymmetric linkages, linkers are required. Technical problems; not serious.
Restarting BLP2 cell, page 111. And the conclusion one, “This doesn’t look promising,” but conclusion two is, “Well, well, looks like this actually all correctly smaller, except #24 and possibly 30 which is shorter, and possibly 13 which is shorter still.” Nothing particularly remarkable preparing fragments, inserting them into dup-del. [00:30:00]
Thoughts on the 3’ fragment on page 119, Sunday, November 19th, with maps. Partial digests with the ACE clone, Tuesday November 20th, page 123 with a rather pretty gel on page 122 of the progressive digestion. But with the conclusion that, “The result is interesting. Very little time effect, and the high-yield of single cut.” [00:31:00] Et cetera.
Some new ideas on duplication, Tuesday, November 20th, page 125, better test of the principle than my first idea which was promoter-promoter-neo, and versus promoter-neo-promoter-neo. It’s to make a construct with loxP sites, et cetera. But I don’t, whether I ever actually did anything with it.
Working on Thanksgiving [00:32:00] Day, Thursday, November 23rd. Results on Sunday, November 26th, page 133, changing BSA to an Xho site, and surprisingly good; looks like 10 out of 48 are correct, one out of 48 is incorrect, and 37 out of 48 are starting material. So I don’t know how those numbers bounce, but anyway, [00:33:00] proceed with #8 for preparative purposes. That is done on the next page, for the 3’ fragment, page 135, Monday November 27th.
Tuesday, November 27th, page 139, “Final, hopefully, assembly.” Ligating, ligation and transformation. [00:34:00] Conclusion, “I never had a better ligation. Hope the Bam worked!!” And final conclusion there, to conclusion two, “Beautiful yield, also 11 out of 24 were good. Streak out #15.” That’s delta — that’s D’138 ACE 12T delta, 12T, the exon, the testis exon of ACE.
Thinking about duplicating the beta-S gene. [00:35:00] Thursday, November 30th, page 141, thinking about beta-S duplication with Nobuyo and Jada Lewis. Trying to get a double copy of beta-S into, to make a model of sickle cell. We never did succeed in doing that. We made good models of thalassemia, but not of sickle cell. Page 143, back from Iguana Island, and about three and a half thousand miles [00:36:00] in 756XP with Roger on December 13th. I don’t remember that trip, oddly enough. So, with Wednesday, December 13th, a check on D’138, ACE 12T, the testis exon delta, with a map. Wednesday, December 13th, starting on these ideas with the loxP sites in loxP site, P-delta, P-neo, and P-neo, loxP-neo, [00:37:00] et cetera, et cetera. Thinking about what to do, and a scheme for making them on the following page. [00:38:00]
Making various fragments, Wednesday, December 20th, page 157. KH21-RSR, and PMC-1-neo poly-single-Pst, et cetera, et cetera. Trying to put in a neo-3’ with a loxP site, and a cozac sequencing in front of neo on page 159, Thursday, December 21st. “Excellent ligation.” [00:39:00] Too good. Maybe something was wrong.
So here we are, getting to the end of the book, Friday the 22nd, page 161, repeat of the Pst partial with calf intestinal alkaline phosphatase, necessary steps for what I hoped to do, ending on Monday, December 25th, Christmas Day. “Proceed process D’161 minis, 1-24. Checked,” et cetera. Conclusion: “#15 is the best candidate, but there are some others. So try one,” [00:40:00] but the same conclusion was, the second gel was, “Lost my bet on E’1. The longer one is probably from the second,” et cetera, et cetera, problem. And that ends the Book D’.