Oliver Smithies:

[00:00:00] So here is beginning of capital S’ book May 10th, 2005 ending in March of 2006.  Starting with continuing of the constructs step six Tuesday, May 10th, page one.  Step seven continued.  Step seven on page five and continued on page seven.  Step eight on page nine.  Various steps needed to make a  construct.  [00:01:00] Wednesday, June 1st, page 15 final step without pDC184 and with pDC215 insulators.  That’s without is DC184 and with is DC215 with the insulators or without.  So here’s Wednesday, June 1st, page 15.  S15 sequences CYP215.  So there are two sequences.  pDC184 is without the insulators and [00:02:00] pDC215 is with the insulators as shown in a diagram on the top of that page.  And on the left-hand side are the two clones, 184 is clone 14 and 215 is clone 37.

And the sequence checks are on two or three pages down.  And all sequences were excellent and match what was expected.  Starting work on FcRn.  The fetal heavy chain [00:03:00] receptor.  Fc receptor.  IgG.  Fc receptor.  Gene.  FcRn.  Neonatal again.  FcRn.  Kumar’s work with the fluorescent probes is beginning to — with the fluorescent markers — is beginning to have positive result.  On page 23 Saturday, July 2nd cardiac hypertrophy markers and some quite nice examples of the [00:04:00] turning on of genes with aortic banding.  Really very good result which led to a publication.

So this is looking at the results again on page 23.  And the paper that it led to was Kumar Pandya, Hyung-Suk Kim, and myself.  “Fibrosis, not cell size, delineates beta-myosin heavy chain reexpression during cardiac hypertrophy.”  And that has some pretty images including actually exactly the one on the left-hand side of — on the right-hand side of page 22.  [00:05:00] That image is reproduced in slightly different color rendition.  But it’s part of Figure 6A in which two cells expressing beta-myosin heavy chain are clearly separated and clearly heavily expressing.

So the comment in my book is that Kumar’s YFP-beta-MHC fusion marks hypertrophy well and follows beta-MHC gene expression well.  The two are correlated as shown on the left-hand side.  [00:06:00] Question whether we could determine whether the flip cells have a different chromatin pattern.  And Kumar began to think about that sort of thing.  Though an interesting remark that Kumar tells me that nearly all of the cells are bigger in the hypertrophied animal whether they’re yellow or not.

[00:07:00] Fairly long break here July — well, not very long.  July 17th to June 23rd.  A visit to UK and celebrating birthday with Roger, who was unfortunately already not very well.  So Thursday, June 23rd the birthday, eightieth.  No experiments.  Writing a grant and reviewing papers.  But four VFR approaches in two hours.  So I’m still flying seven five six X-ray Popper GPS two seven and ILS six with a procedure turn at Burlington and [00:08:00] GPS, etc.  Two IGX with eight four Kilo Sierra.  So one with — flew both seven five six X-ray Popper the 182 and eight four Kilo Sierra the motor glider.  Actually I was not incorrect.  We did not visit on the eightieth birthday.  Visit was later to celebrate.  Not actually on the birthday.  [00:09:00] Progress report completed Friday, July 1st.  Cardiac hypertrophy markers are doing well.

Next entry Saturday, July 2nd, page 27.  Thoughts on ANF with quite a big insert on the left-hand side pDC143.  The construct without insulators.  [00:10:00] Worrying about the complexity of different plans on Saturday, July 2nd, page 27.  The Q’157 plan for making ANF is too complex.  Better try an improved MCAD type upstream duplication, etc., with a single color gene and various other thoughts.  [00:11:00] Saturday, July 23rd.  Thinking about publications.  Possible language.  We propose a testable hypothesis that hypertrophic stimuli can invert a developmental heterochromatization of some heart-related portions of the genome.  One consequence is a highly stable or metastable or unstable [00:12:00] cell-autonomous switching, etc., etc.  Trying to think about chromatin changes.  Check with a literature search.  Looks a bit premature.  Too many others have described on-off switching.  Need to prove reversibility or not.  [00:13:00] pDC143 modification as a step to further experiment.  Construct targeting for the ANF locus.  Or no, with an ANF promoter.  Long homology 10.8.  Let’s forget about that, it’s not important.  Typical review of status Thursday [00:14:00] through Friday, August 25th through 26th.  And six things that I have, I have, I have, I have, I need, and I have.  Various things.  And assembly steps.  Doing what was necessary.  Typical pages.  [00:15:00]

And paper on the bradykinin receptor absence on page 67 Tuesday, 13th near final, with some comments of Seigo and Masao and a conclusion.  It demonstrated that absence of bradykinin B2 receptor in Akita diabetic mice increases oxidative stress.  Mitochondrial turnover.  Mitochondrial DNA mutation.  Etc., etc.  These findings lead us to suggest that premature senescence underlies many of the [00:16:00] complications of diabetes and that bradykinin plays an important role in preventing them.  Still making constructs though, for many pages still.  With a typical final assembly on Wednesday, September 28th page 83.  For a PGTK driving along 5’ promoter for driving cyan.  [00:17:00] From Tuesday September 27th page 85 the switching business.  First evidence of cyan latched off in transaortic coarctation animal.  Where there are many cells that don’t have the nuclear cyan and some cells that do, with a comment that this could be attack response as the green nuclei are more in fibrotic areas.  [00:18:00] So it looks like it might be related to fibrosis.  Friday November 4th page 95.  A new targeting project which is really basically Nobuyo’s is to [00:19:00] use, look at the mitochondrial DNA synthase, the POLg locus which codes for mitochondrial DNA polymerase gamma.  And that a mutation in that which removes the editing function has very much an aging type phenotype.  So Nobuyo has written a grant containing that type of material which we did go on very successfully to — or she did with Ray Fox very successfully reach a conclusion.  [00:20:00] Thinking about publishing Kumar’s TAC results Monday, December 5th, page 99.  Possible title:  Correlated but not concordant stochastic on-off switching of genes during development and under stress.  [00:21:00] With a rather interesting thought that was persistent later that Nobuyo suggested that the half intensity blue cells that were sometimes seen are due to LBG insulator AC cyanate cyan being switched off in one nucleus but not the other cell, other cardiomyocyte.  In some images that show on page 98 show cells that appear to be half intensity.  [00:22:00] Some work on testis ACE again on page 101 Friday, December 16th conversion of testis ACE-CRE testis ACE F1C recombinase.  Flip recombinase.  Wanting to have another control besides loxP.  [00:23:00] So OSDupDel testis was being considered.  A very complicated construct.  [00:24:00] For about the nth time a revised list of steps.  Thursday, December 29th, page 111.  Going towards one of the constructs with FRT and loxP.  [00:25:00] Step one.  S’115.  Step two.  S’117.  Step three.  S’119.  Etc.  All the steps there are being listed that were being documented, being planned.  On Thursday, December 29th, page 111.  So if we turn to page 115 we see step one has been achieved.  And we turn to page 117.  Step two has been carried out.  Etc.  Step three January 11th, page 119.  Step four 121.  Step six, page 123.   [00:26:00]

So Monday January 23rd page 113 now in 2006.  Three steps to assemble OSDupDel testis.  It’s complicated construct with [00:27:00] FLPs and loxs, where the map is shown.  Final assembly step four Monday January 30th page 141.  OSDupDel testis.  [00:28:00] Friday February 3rd page 143.  Cora-Jean’s human vascular endothelial cells cuvettes plus or minus ethidium bromide plus or minus uridine.  Trying to get cells that don’t have mitochondria in them.  About a month in culture.  Very promising for loss of mitochondria.  [00:29:00] Control and ethidium bromide.  And ethidium bromide plus uridine images.  [00:30:00] More images from Kumar page 151, and page 153 and 154.  [00:31:00] Going back to retrieve all the data that had been done on cell switching page 157 Tuesday February 28th.  Compilation of all the experiments that were relevant.  Note listed on the left-hand side on page 156.  Starting June 1998 thinking of cell type switching and renin production.  Going on in different ways.  [00:32:00] For example May 2003 John and Joe making long beta-MHC promoter construct with insulator.  And February 2006 Kumar’s Y beta and Ren TG that distributions in survival with LG insulator AC TAC, etc.  So a list of all that had been done and thought about that problem.  Need some more work on bradykinin B2 receptor experiment.  Conditional knockout of B2 and B1.  Need some upstream DNA and how to get it.  [00:33:00] So primers are being — tried to make primers.  Tried to make probes using new primers.  Last page 163 Saturday, March 11th and page 162, with different primers for different probes.  A list of the different ones on page 162.  That’s the end of book S’.