Oliver Smithies:[00:00:00] So here is book rho, beginning May 24th, 1989 now, listed with address, O. Smithies Department of Pathology, University of North Carolina Chapel Hill, and goes from May through October of the same year. And interesting, this period, I don’t remember these experiments very well, but I do remember the feeling that the move from Wisconsin to Chapel Hill, although altogether an enjoyable move, nonetheless was traumatic in some ways, because of change in surroundings, et cetera, and it took me about six months before, [00:01:00] as I remember saying it, at least to some people, that I got my brain back. I didn’t seem to have any new ideas for a while, so it may be, that’s why I don’t remember this work very well, and I don’t think it ever led to anything very useful.
But anyway, starting on page 1, ligating the 2.1kb HindIII DraI fragment, Xba159 into the beta — what am I ligating it into? Let’s see. Into rho-1, pi-105 supernatant. [00:02:00] We’ll hope for more.
Here, this is the beginning of book rho, page 1 and ligating in the 2.1kb HindIII Dra1 c-kit fragment into a cloning vector. [00:03:00]
And continuing with the construct on page 3, and page 5. [00:04:00] And now using a different vector on page 7, Friday, May 26th, pUC118, and EcoRI HindIII vector. Got this from Terry — I’m not just sure which Terry that is. I know Terry Magnusson, but I think it’s before Terry’s time.
Terry Smith. Who was Terry Smith?
Irish, Irish guy.
Where was he — Terry. So this is pUC119 from Terry Smith, he was [00:05:00] Nobuyo’s postdoc.
I’m sure it is, yeah.
Various DNA constructs, and electrophoresis of the electrophoretic tests. Rho11, HindIII, plus P-pi delta fragment, et cetera.
An interesting comment on Sunday, May 28th, the rho ligation, that’s transforming with different amounts of material, with a comment, “Mistake. Never plate everything the first time. [00:06:00] If you’re doing a dose response, leave at least a third so here is 50μL, 100μL, 200, 400, and the rest, and better to just stop at 400 and kept the rest, but couldn’t repeat.” In fact, it’s a general technique I’ve since used: never use more than two-thirds of anything that you might have to repeat. Better use a third, and then you have enough for two repeats.
Memorial Day, May 29th, Monday, thinking about my father and mother, and sister Nancy. Trying to ligate the [00:07:00] Dra/HindIII 2.1 fragment into pU1C. And so on, continuing the constructs over the next days. [00:08:00]
So trying to use a FLAG sequence, tests of a leader/FLAG series on Sunday, June 4th, page 29. And Nobuyo sequenced these [00:09:00] plasmids. Rho21 — Rho29 #21, and Rho21 #1 were sequenced. And so, conclusion that, “Rho29 #21 can be used, and Rho21 #2 was OK on both.” The sequencing.
More sequences now, June 10th, beginning to do more of that type of work, though these were [00:10:00] Nobuyo’s hand sequencing, not by any machine.
So I went abroad to a meeting between June 10th and July 5th, and it was a very luxurious meeting as I remember in France with a large amount of food and drink, and I came back on the Concorde, but I regretted having that opportunity only because I couldn’t enjoy the food and drink in the Concorde; I was saturated with it, but I came back Mach-2 at 56,500 feet at [00:11:00] -58 degrees Centigrade at 1,320 miles per hour, Mach-2, in the Concorde, so I did get to fly it. I’d try it again, but never managed to get another chance. It’s a neat entry on page 35, Wednesday, July 5th. And there are, looking at the construct, which is lead, FLAG, c-kit, terminator, terminator, not Hind, so. It’s a FLAG sequence associated with c-kit. [00:12:00]
And characterizing plasmids and constructs continues. On pages and pages, working on making plasmids. And here is a map, a large mini preparation, Monday July 10th, [00:13:00] page 57, with a map of Rho57 mini #22 pUC118, EcoRI leader FLAG sequence c-kit, so it’s still working on trying to express a c-kit.[00:14:00] Nobuyo – was it Stuart Bentley? Was there another Bentley? Hmm? Because I don’t know why I’m using his surname.
Well I don’t know why I’m using his surname – suggests that Bentley work on the — and Bentley’s later data, not Stuart. Did we call him Bentley, not Stuart?
Stuart, I don’t know Oliver.
You don’t remember, I don’t remember.
He was here. I don’t know what stage…
He worked with steel, anyway, we’ll go on. OK Jenny, are we alive?
OS:[00:15:00] Some notes on what I hope the steel factor will do, can be used to increase germline transmission, useful for getting gene correction in bone marrow cells. So, in a normal animal, there’s very few stem cells being produced in the bone marrow, most of the progeny are differentiated. The hope that steel factor will keep the primordial stem cell growing so that I get more primordial bone cells using steel. [00:16:00]
So trying to get Sal-HindIII fragment with a leader, FLAG sequence, and c-kit on it, a Sal HindIII fragment on page 57, Sunday, July 16th. More digests of L/F c-kit, leader/FLAG c-kit, and prep gels, and so on. [00:17:00]
Saturday, July 22nd, page 85, “Hoping that these are the final ligations!” c-kit, not Sal to p284, kit, not Sal, and of c-kit HindIII Sal into pH-beta, not kit — not Sal-kit. Both ligations are good. [00:18:00] And so, we use DH-5alpha-F1, on Rho-49, and picked 12 minis, one of the ligations and 12 of the other mini digests.
So, page 89, Thursday, July 27th, page 88 says that, “See later note, all except #9 were correct,” and that’s for p284 and pH-beta, all were correct. [00:19:00] Continuing many pages more.
Rho91 super mini #15, let’s call it a-c-kit, meaning human beta-actin promoter, driving c-kit. This is p-Rho91 a-c-kit, actin-driven c-kit. [00:20:00]
Comment, “The problems of isolating clones and proceeding without sequences,” note on Wednesday, August 16th, page 117 and the facing page, 116, [00:21:00] that “Dana sequenced the c-kit used for these constructions after finding the protein was too short. There’s a two base pair deletion, and some point mutations in the coding region, so I have to repeat. Critical things need to be sequenced.”
Now, we’re talking about HPRT+ going to HPRT-, the rationale “98-12 has been successful in going to the germline recently, but it’s awkward, because it’s now HPRT+. I need a deletion mutant, HPRT-, from it, that mimics [00:22:00] EK, but has better clonal history.
So, on Tuesday, September 5th, page 119 is the comment, “The germline transmission paper after PNAS,” that is the germline transmission of correction of HPRT- to HPRT+, and I will check that reference. So Tuesday, September 5th, page 119.
So, Tuesday, September 5th, a note [00:23:00] on page 119 that the paper showing the germline transmission of a corrected ES cell mutant, can go germline, was submitted. This is a paper by Bev Koller, Lora Hagemann, Tom Doetschman, John Hagaman, Shu Huang, Phillip Williams, Neal First, Nobuyo Maeda, and Oliver Smithies. This is our first germline transmission of a gene that was changed, so Bev Koller was the first author of this paper published in November ’89 in PNAS. So that was a happy day. Thinking about that, [00:24:00] though, on the opposite page, that clone 1998, 8-12, the HPRT+ clone has been successful in going germline, but it’s awkward because it’s now HPRT+, need a deletion mutant with a better history that can be used to go from HPRT- to HPRT+. And some thoughts on what to do about that. [00:25:00]
So, proceeding with these new ideas, and putting them into plasmids. The real start is, said in double circle, page 127, Thursday September 7th, “The real start is starting with pNMR122-rho121,” that’s your plasmid, Nobuyo, isn’t it? You remember that plasmid?
Maybe. I don’t remember anything about it.
No, I think it’s your plasmid, NMR121, starting with Nobuyo’s plasmid, and — cutting it with BstX1, [00:26:00] and blunt-ending it, et cetera. And, ligating in BstI, BamHI-containing exon 3, of the HPRT gene. [00:27:00]
So, Rho-133 minis at the potassium acetate stage, on page 141, Sunday, September 10th, where about 24 different minis were looked at. The conclusion, “A good selection. And Nobuyo Maeda says, the dimer, or nick circle band migrates close to the double-stranded DNA,” et cetera, “and looking, 4, 6, and 16 look promising. Need a 7.7kb fragment.” Conclusion: “All three are good, 4, 6, and 16, all three [00:28:00] are good, but 16 is a trace larger, better check some more digests. Character completion, but the OD was a bit disappointing.”
So here we are on Monday, September 11th, checking these clones with Pst-Xho, Pst-Eco, Pst-HindIII, trying to see if the exon 3 is there. [00:29:00]
So, back to 98-12, with Wednesday, September 13th, page 147, “Help from Nobuyo, she ran the gel post-complete digestion, and Sylvia repaired bulk DNA from Rho133.6, which was renamed now Rho143.” [00:30:00] And a map of Rho57 mini 22 after sequencing. Here is a comment on Saturday, October 14th, that “Dana tested H-beta-kit and/or 284-kit and found too short a product, and revealed a frameshift mutation that was commented on earlier.” So here is correction of c-kit planned on page 149 [00:31:00]
which is continued then on the following pages.
So the book ends with a review of the stocks on [00:32:00] Thursday, October 19th, “Material needed for recloning into the two expression vectors are, 1) the corrected equivalent of Rho57 mini 22, that’s with the c-kit corrected, need a FLAG-c-kit prepared from it on a Sal-HindIII fragment, and a Sal-Not fragment, then putting it into p284, and pH-beta CPR1, so plans to recover the mistakes of the previous cloning efforts. And that ends book rho.