Oliver Smithies:[00:00:00] So this is Book P’, starting June 25th, 2003, and ending December of the same year, so about six months, beginning with construct-making.
Nothing very remarkable in the first 20 or so pages. [00:01:00] Looking at the possible vectors, transgene vectors, Tuesday, July 8th, page 21. “For the cell-type switching, we’ve been using HPRT promoter region. HPRT Exon 3, ANF‑3’, beta-MHC‑3’’. The transient pairs BG.AC, etc., have had problem.” With some thoughts, that are not really very illuminating. [00:02:00] And Tuesday, July 8th, page 23, continuing the colonies that were being looked at or cultures being made by Kim, Ray, and Randy. Various images. Friday, July 11th, page 29, “Dominic needs data on ±2 insulators on paired genes.” So the computer files made of [00:03:00] RIIRRAVR and BIIBRAVR, that are different constructs. (pause)
Progress report due. So some cell experiments reported, of blue and cyan colonies — [00:04:00] of discrimination between cells giving blue, green, and blue-plus-green cells beating, with beta‑MHC driving green and alpha‑MHC driving the cyan. So some blue cytoplasm, green nucleus cells and some where they’re either blue alone or green alone, in the progress report‑ “The best apparent discrimination that we’ve had yet” — with some images.[00:05:00] Friday, July 11th, page 33, thinking about making different constructs. And then, on the left-hand side, the comment, “Too complicated. Quit.” (long pause) [00:06:00] (pause) I wanted to turn RIIR around, Friday, July 18th, page 45. (pause) Still [00:07:00] struggling with these constructs. (long pause) Pages and pages of constructs. Some thoughts on promoter interference and on insulators, Wednesday, August 13th, page 75. “Maybe competition for enhancers is taking place.” [00:08:00] With five thoughts. (pause) Just making constructs and making constructs — (pause) with nothing remarkable. [00:09:00] (long pause)
Getting deeper into it, on Thursday, November 20th, page 119. Had given the Berryhill lecture — and several papers [00:10:00] finished and back to work, now thinking about MCAD, which is the medium-chain acyl-CoA dehydrogenase promoter — which is active in cells that are myocytes, I believe. Page 119, Thursday, November 20th, beginning to think about using the gene coding for medium-chain acyl‑CoA dehydrogenase [00:11:00] coupled with cytoplasmic cyan, to look for switching of cells, since, when the adult-to-fetal shift occurs during hypertrophy, the cells revert to the fetal stage of not using fat for their energy sources. So an element that can be used for looking at the adult-to-fetal switch, the MCAD promoter. So beginning that work in the following few pages.
Monday, December [00:12:00] 1st, page 127, targeting inserts in control for the MCAD. So trying to make the plasmid. (pause)
This plan is discussed a little on Friday, December 5th, page 131. “Since the main aim is to get the MCAD promoter-driven expression of CFP and the secondary aim is to preserve the wild-type gene function, it seems best to put [00:13:00] CFP upstream,” etc., etc. So on the next page, on page 132, has five kilobases upstream.
As ever, new strategies, Friday, December 12th, page 137, this time Nobuyo’s.
Have better PCR conditions, with a hot start, on Saturday, December 13th, page 139, which became later my standard method, a hot start. “With good tips,” it says. [00:14:00] PCR, page 139. (pause)
With, page 147, declares targeting construct, being Saturday, December 20th, the targeting construct being listed. And then, on the opposite page, December 12th to 18th, a review of the status of the animal — of the various animal. “Valuable comparison.” LGB ANF, LBG [00:15:00] HPRT. That’s LBG at the ANF locus and LBG at the HPRT locus. BG.AC A‑mito‑C at the HPRT… And long BG… etc. “The cells are available.” And whether any mice… (pause) Christmas Eve, some phage tests, Wednesday, December 24, page 153. And Christmas Day. [00:16:00] Looking for a probe — 300-base pair mini probe. (pause) Back, on Tuesday, January 6, from a visit to Roger, in London. [00:17:00] And the book ends with purifying “669,” that plasma, a fragment needed for further constructs. So ends Book P’. [00:17:35]