Oliver Smithies:[00:00:00] So this is the start of Book O’, beginning February 28th, 2003, and going through June 23rd of 2003. Starting off with the addition of cyan markers to an existing plasmid, Friday, February 28th, page 1. [00:01:00]
And that construct work was going on, still Friday, March 14th, page 19, a cleanup of a fragment that was needed. Friday, March 14th, page 21, first test of GFR vs. albumin urea, testing the hypothesis that as GFR decreases, albumin urea should increase. This work was done with Nobuyuki Takahashi in [00:02:00] conjunction with Bill Arendshorst, help in Bill’s lab. Two rats were tested by varying the clamp of the renal arteries so that the blood pressure decreased and was expected to lower the GFR. But, the result was, in the excretion, in each rat, the excretion of albumin in micrograms per minute by ELISA decreased as the GFR decreased. [00:03:00] And with a conclusion that I needed to decrease the GFR more while maintaining sufficient urine flow, etc. February 2007, the GFR was measured by continuous inulin, a label injection. So, the gels show really no significant increase, but rather a decrease in albumin.
Back to constructs, the following day, following Monday, March 17th, page 23. [00:04:00] Monday, March 24th, beginning to help Joe Brackham, having trouble with pSKB1-beta-actin, HRGN parlay, should be nuclear green, with some photographs of, or images of cells that had nuclear fluorescence. [00:05:00] Further imaging on the next pages, intermingled with constructs. So that’s [00:06:00] with experiments with pSKB1 type of experiment, inserting a single copy into the HPRT locus. A new plan, Saturday March 29, “Since HIG is not yet working, I’ll try Dominic’s pSKB1 with a short beta-actin promoter. I had a very encouraging email from Ananth Karumanchi [00:07:00] on Tuesday the 25th of March, which he’s saying, but he read my PNAS paper, and thinks that he understands now his own experiments in which albumin urea was obtained in rats by adenovirus. Particularly, he says, ‘I think that your hypothesis [00:08:00] might explain the mechanisms of albumin urea in patients with pre-eclampsia. Everyone has focused on the size selectivity of the slit diaphragm, and GBM, but your hypothesis is more elegant, and likely be true. I really loved your paper.’ So, I talked to him after receiving his email, where he had used adenovirus infecting the liver to secrete soluble Flt-1, the soluble Flt-1 being the soluble form of the VEGF receptor. And that this causes a loss of endothelial fenestrations, but no changes in the GBM or in podocytes, yet with massive albumin urea. [00:09:00] And he will send us the virus, and we will measure plasma and urinary albumin, and determine GFR. And he does not know this, but we expect very severe decrease in GFR. Another example!! Also very related to eclampsia, since the placenta secretes more S-Flt-1 in this condition.” In other words, beginning on this day to think about working again, perhaps working with pre-eclampsia, remembering that pre-eclampsia was the subject that John Krege had been working on when he introduced me to [00:10:00] blood pressure work. So here is Ananth Karumanchi working on pre-eclampsia, getting me to think more about what’s happening in the kidney. But no experiments immediately.
So Tuesday, April 1st, a plan of how to make Dominic’s version of a short beta-actin driven HRGN. More constructs over the next pages. More images of fluorescent cells on page 53, Wednesday April 2nd, the following page. Joe’s colonies, which had his colonies C, was now growing up sufficiently to give beating cardiomyocytes, [00:11:00] three or four beating areas found, but none are fluorescent with either yellow or blue. So these initial experiments Joe had obtained beating cells, but no fluorescence. So that part of the experiment hadn’t worked. It did, later.
Every now and then a new plan, Friday April 4th, Nobuyo Maeda’s birthday, to subclone beta-actin, etc. etc. A new plan. Pages [00:12:00] more of constructs. Assembly with pSKB1 of short beta-actin promoter with g-nuclear and short beta-actin, and HoxB4 cyan, complicated [mix?] on page 77, Tuesday, April 15th. [00:13:00]
Looking at colonies on Tuesday, April 22nd, presumably Joe Brackham’s but not stated, so plenty of cells showing nuclear fluorescence with cyan, but nearly all, or all are feeder cells. More images on the following several pages. For example, Sunday, April [00:14:00] 27th, page 93, the culture’s been going for six days, and some of the colonies are growing very well, although all show signs of being mixed.
Thursday April 24th, page 99, urine checks. But Nobuyuki found that approximately normal samples collected from Sprague Dawley and Wistar Kyoto, wild type rats, that the Sprague Dawley have pathological levels of albumin before anything is done, [00:15:00] as they did in the O’21 experiment, whereas Wistar Kyoto are fine, they don’t have albumin urea. So they’re better to use.
Long beta-myosin heavy chain promoter tests being attempted on page 105, Friday, May 2nd, long beta-MHC promoter LBG with an insulator, and then AC at the HPRT locus. [00:16:00] [00:17:00]
Saturday, May 24th, page 123, “You can’t see any colonies, can’t find the good colony from yesterday.” But May 25th, also no significant differences, but there were some paired nuclei observed, suggesting that they might be cardiomyocytes.
Pages of constructs, [00:18:00] nothing particularly remarkable. With the usual sort of interim result, page 145, Friday June 6th, “Various rescues!!!” With worrying on Thursday, June 12th, page 147, of what many people must have worried about, the complexity of transcription factors, and their cross-reactions [00:19:00] are very strange. “Suppose therefore, that it is not rational, but rather adventitious, just accidental. Evolution can change single-gene DNA very rapidly, but cannot change multiple systems already in use. Therefore, consider the possibility of accidental clustering of factors.” Which I called, “opportunistic evolution in complex genomes.” [00:20:00] With a comment from Kumar that, “Promoter regions of the alpha and beta-MHC are very different, but the coding sequences are very alike.”
More images of cells, but nothing very convincing, or leading to any real conclusions. [00:21:00] Tried to think about a model for Nobuyo’s apoE trapping ideas, Monday, June 16th. It didn’t go anywhere.
Monday, June 23rd, my and Roger’s 78th birthday, insulators into a previous construct. Insulators, with helping Dominic. [00:22:00] And the book ends with more images of cells, but with no particular important conclusion on Tuesday, July 1st.