Oliver Smithies:

[00:00:00] So this is the beginning book L’, dating from April 5th, 2001 to July of the same year.  It continues initially with construction of plasmids which would cause the expression of various colored fluorescent proteins when inserted into the HPRT locus using the plasmid pSkb1 code test pol-A, which had been constructed earlier.  So a retransformation of a couple [00:01:00] of them on page 1, April 1st.

Some discussion on the following pages, page 5, Tuesday April 10th, of blocking the NKCC2 channel.  That’s the sodium-potassium exchanging channel in the kidney with diagrams of what was being thought of on page 4.

Continuing then with checks on the colored proteins, fluorescent protein construct [00:02:00] in the next few pages.  And Wednesday, April 25th, considering a hypothesis that cells might be able to exist in two states, which had been the subject of a grant application.  In other words, if a stimulus was applied, a cell might switch from a low-expressing state to a high-expressing state, and be maintained in that state, even when the stimulus or the stress was reduced, because of inability to switch back until the stimulus was much lower on the return journey as it were on the forward journey, historesis effect.  And, so [00:03:00] this is the discussion of the pathways involved.

And with some remarks on Sunday, May 5th, quite a bit later of Loren Field using alpha-MHC-neo selection to induce or select myocytes in ES cell cultures, a possibility that the switching had occurred.  So, continue with the fluorescent proteins again, again, page 13, [00:04:00] now looking at the actual colonies on page 15 and one can see the difference in fluorescence, that it was not uniform everywhere, and so HRGN is expected to be in the nucleus, but some variation in staining, or in fluorescence was observed, and is talked about, or looked at in the next few pages. [00:05:00]

Continuing to work on these different fluorescent constructs in the next few pages, and changing, for example, on page 27, on Wednesday, May 9th, changing the nuclear expression of the cyan fluorescent protein, to just non-nuclear expression.

Trying to have the ability to control the fluorescent protein with a different promoter, and specifically with the beta-myosin heavy chain [00:06:00] promoter, this work begun on page 35, Thursday, May 17th, having received a plasmid from Fadia Haddad that has the beta-MHC promoter.  There is a good diagram on that page, of what I’m attempting to do.  Beta-actin promoter and changing to the beta-MHC promoter is the idea, in HRGN.

Some more fluorescent cells being looked at on page 39, [00:07:00] Tuesday, May 22nd.  And continuing in a similar vein on Wednesday, May 23rd, page 45, where everyone can see the comments attached to the images.  In here about the wavelengths of the particular absorption and emission spectrum, and relative intensities on page 47, Friday, May 25th.  [00:08:00] Continuing now with the beta-MHC promoter into a plasmid as it was obtained commercially, GL3, on Saturday, May 26th.  Sequencing of a plasmid derived from pSKB1, with the beta-actin promoter on page 55, Sunday, May 27th. [00:09:00] With an error being detected.  So having to redesign the promoter construct on the following page, Sunday May 27th, page 57.  Again, some problems in the logic, that were later discovered.  And all these being reviewed on page 59, Monday May 28th.

And so on, continuing then in the following pages, looking at colonies and [00:10:00] continuing to make constructs for these various fluorescent proteins.  And having to start again every now and then, as usual, Tuesday, June 12th, page 77, a new start for pSKB1 AAT echo, but it was canceled.  And with the usual again, again, on page 79, Tuesday, June 12th. [00:11:00]

Beginning to think about cardiomyocytes derived from ES cells on page 81, Tuesday, June 12th.  Joe Brackham has been following cultures of cloned ES cells with HRGN and in the HPRT locus using our construct pSKB1 code test reverse pol-A.  And, the nuclear fluorescence is very cell-type dependent, as it’s beautifully strong in beating myocytes, so that we are ready to show that the myocytes were beating and fluorescent, and although this is not a video in the book, page 81 [00:12:00] at the bottom says, the cardiomyocytes are clearly binuclear; that is, they’re post-14 day and the bright cells were beating, with an image of the bright cells, and of the phase contrast on page 80, so at least the HRGN could give rise to beating cardiomyocyte, with more pictures, more images on page 82 and 83, and 84.  One particularly beautiful nuclear fluorescence on page 84.

A whole summary [00:13:00] of where what’s being made, and what’s being considered on pages 88 and 89, Friday, June 15th, under the title “more thoughts” of all of the different promoters, and constructs.  Eventually this fluorescence of cardiomyocytes led to apublication considerably later with Kumar Pandya. [00:14:00]

Friday June 22nd, page 101, “Backup for step 1, and again to the nth power.  Always repeating.” (laughter) Wednesday June 27th, page 111, “Back to go,” etc., “Step 1 again, but it was canceled because of an interpretive error.” [00:15:00] Nothing very remarkable in this part of the book, just slog, slog, slog.  Redesigning, Friday July 6th, page 125, with steps A, B, C, D, E, F.

Back to the idea of switching and some developing of computer simulation of this on page 137, Thursday, July 12, further switching program idea [00:16:00] with some thoughts that if you can have switching just between two states and there are many of these switches, diversity can be obtained very easily.  But there is some sort of auto-feedback necessary at the threshold to keep the model stable in one of its two states.  And, with some thoughts that current ability to obtain cardiomyocytes from embryonic stem cells suggests that there are really only a few steps needed to get to different types of cells. [00:17:00] And again, hematopoietic stem cells to cardiomyocytes also suggest the same, a few steps.  And may be able to make a beta-MHC puro, etc., animal, and switch to hematopoietic stem cell, switching a hematopoietic stem cell to a cardiomyocyte, the idea.  On page 136, “The decisions must be self-perpetuating, or some self-feedback.”  This is one of my overnight little pieces of paper. [00:18:00] For example, if there was two possible switches, two possible states of 10 switches, you could get 1,000 different cell types with the different combinations.  Not very profound thoughts, but it was stimulating to me to think about the switching.

I got into the habit of assembling sequences in the computer before trying to assemble them in the test tube, and page 145, Wednesday, July 18th, comments [00:19:00] on this, making of A-MHC alpha-myosin-heavy chain, neo alpha-myosin heavy chain cyan, following several steps scheme assembled in the computer, with the aim to take beta-MHC, HPRT-plus, and go to beta-MHC, AMHC, etc. etc., different constructs.  But it was a useful thing to go assemble in the computer first, then making sure that it would assemble better in experiments. [00:20:00] Avoid making silly mistakes.

Four-piece assembly being considered on page 147, Thursday, July 19th, with the diagram, in order to make an MHC-omega construct.

Starting with a three-fragment assembly on page 149, the following page, [00:21:00] Thursday, July 19th.  I think to remember, the record was five fragments.  And starting on page 157, July 24th, Tuesday, on making this NAAC-omega construct, which had been diagrammed a little earlier. [00:22:00]

Just stop it a minute.

So this is resuming at page 157, Tuesday, July 24th, start of making a new plasmid, which was called, “NAAC-omega,” had been discussed earlier, probably in relation to omega, making a stable hematopoietic stem cell type of construct, although a little unclear.

The book ends [00:23:00] on page 159, Wednesday, July 25th, with just continuing making different constructs.  And so that’s the book L’.