Oliver Smithies:[00:00:00] This is the beginning of book Greek omega beginning on February 4th, 1993 and ending June of the same year. The initial experiments are continuation of devising the tools in order to isolate construct enabling modification of the angiotensin-converting enzyme gene. In particular on page one, Thursday, February 4th a probe made for the 5’ part of the ACE gene is being cut into two pieces, one of which was called blue, one of which was called yellow, that can be used to detect [00:01:00] exon 1 or exon 2 sequences. And on the next page Friday, February 5th, page three, work is going on with digesting of previously made construct BHNTK and so on. There’s a summary of the general situation on page five, Wednesday, February 17th. There’s a gap there between February 5th entry and February 17th with a note “Back from the Dutch Lesser Antilles.” This must have been a trip almost certainly with Field Morey and my flying friends to go sailing. We’d fly down there in our airplanes and then [00:02:00] go rent a sailboat and go sailing for a while.
And here it is then. Review of the probes for the 5’ ACE gene and describing what the results are. Most likely an EcoRI fragment 11-kilobase and HindIII fragment are what is being required. [00:03:00] Now on the following page Thursday, February 18th, page seven, beginning to work on getting the 3’ ACE gene now using the same technique to develop a better probe, etc. Nothing very surprising, just straightforward work of isolating the DNA needed to make constructs. Still thinking about this idea of not altering the frequency of recombination on page nine, February 19th. Kinasing a splinker/ligation with this idea to block the end of the [00:04:00] construct with a piece of DNA that would make a single-stranded cap, quite nicely diagrammed about mid page nine. And this work is continuing in the next few pages. Returning back to ACE gene on Saturday, February 27th, page 13. Just looking to make another primer. And picking phages from 5’ ACE intron on following page 15, Sunday, February 28th. [00:05:00] Several promising but they didn’t survive the secondary pick. More work on 3’ ACE gene probe, page 17. And some thoughts on the previous BHNTK idea on Tuesday, March 2nd, page 19. The idea is to see whether transient expression of TK causes any problems, and suggestively continual use of [00:06:00] GANC with increased enrichment, etc.
Happy result on page 21, Saturday, March 7th with a conclusion which is March 10th, Wednesday. This worked spectacularly, 5’ promoter had many more weaker false positives but also had positives. So this was getting the 3’ last intron and exon 6 and 7 probe. Some good results. [00:07:00] Splinker, the little cap at the end. Work is resumed on Monday, March 8th, page 23 with a conclusion that the closed circle assay worked very well, etc. which is the assay as described on page 23. Continuing with these ideas again on page 27, Wednesday, March 10th. More splinker ideas. [00:08:00] Worrying about whether the procedure is fully valid. Tests of the current splinker DNA page 29, Wednesday, March 10th with a conclusion that the treatment was not complete but that there is some protection from digestion with an exonuclease. The protection provided by the splinker at the end [00:09:00] and proceed. Making more material following page.
And back to ACE again on Wednesday, March 10th, page 33. The 3’ ACE. Trying to get that piece, the diagram of which is shown on page 32. So I’m alternating work on ACE and work on the splinker, more page 35 on the splinker. And yet back on page 37 to Saturday, March 13th to 5’ ACE minis.[00:10:00] And got three candidates being tested. And all three are the same and have an 11-kilobase EcoRI fragment which would later prove to hybridize to the 5’ probe. So these are 5’ ACE minis. More subcloning of them on the following page, 39 and going back to the 3’ subclone page 41. And so forth. Looking at the candidates on page 43, the 5’ ACE candidates. [00:11:00] Not quite sure whether I’ve got what I need, but here are diagrams on the way. And redigestion page 45. Same result. More of the same type of thing on page 47. Retry on 5’ ACE subclone with the conclusion abandon. Make a good phage mini. Prepare an EcoRI digest. Isolate the 11-kb fragment and subclone. Continuing with the work on the following pages. [00:12:00]
Here we go on page 53, Friday, March 27th. Test of omega 3 BamHI digest of BHNTK and with a diagram of BHNTK Bam neo promoter backwards and promoter one two TK and the TK promoter and the neo genes with the neo genes and the TK genes being in the opposite direction from the promoter one two.
Test without ganciclovir and with ganciclovir for the whole time, etc. [00:13:00] So the two orientations of the TK gene being looked at. Trying to see what happens if the TK gene is in the opposite direction. The results are on page 52 and so ganciclovir is having no effect. Changing the number of homologous versus non-homologous recombinants. In other words the experiment is [00:14:00] a negative result. No change in the frequency of homologous recombination versus non-homologous. And testing the reverse orientation of BHNTK on page 5, or getting ready to test that material by making it, but haven’t got the right product yet. So here we are on page 57 Tuesday, April 6th looking at 3’ ACE [00:15:00] sequence on the 7-kilobase EcoRI fragment with a map of the construct in ori vector on page 56. So getting towards having the 3’ ACE fragment correct. Continuing to try to improve in the following pages.[00:16:00] Some interactions with Emma Whitelaw regarding thalassemic mice on page 65 relevant to Barry Whitney’s work, Postdoc Barry Whitney. Still revisiting BHNTK on Saturday, April 24th, page 67. Looking at the possible orientations of the constructs [00:17:00] with a conclusion that things are looking reasonably good, page 66.
Looking at the 11-kilobase 5’ EcoRI fragment, page 71. Still [00:18:00] struggling with the difficulty of seeing any effect of these constructs in changing homologous versus non-homologous recombination. All of those BHNTK constructs all dependent on getting homologous recombination to introduce the promoter and exons 1 and 2 into the deleted HPRT gene by homologous recombination using either E14 ES cells or BK4 was another possibility being considered on page 73. Wednesday, May 12th. So there was the test with BK4 and with no real alteration in the ratio. [00:19:00] Considering possible construct of the ACE gene on page 75, Wednesday, May 12th. Triple fragment ligation being considered. Those multiple ligations with different ends turned out to be very productive. Nobuyo was the first person who started to use them and we used them. Eventually one was made to work with five different fragments all with ends so that [00:20:00] it could only back in one way. But anyway there’s a possible construct on page 75. On page 74 the final will be so on and so forth. With a linearization site Xho in the sequence. And HindIII and EcoRI fragment being used in the construct. HindIII Xho Xho Eco Eco HindIII three fragments. Now the experiment is being [00:21:00] considered further on page 77.
Improvements in the concept being considered on page 83, Friday, May 14th. Redesign. More construct revision Saturday, May 15th. Digest moving towards getting what was needed on the following page starting with pMC1neo poly(A). [00:22:00] Various tests of the constructs. Some new sequences. New primer sequences to change restriction sites for example to knock out HindIII and change it to Xba, knock out Sac and change it to Xba, and knock out Xho and change it to Not, etc. on page 91. It’s a technique that we used a lot, changing sites. [00:23:00] And some of the site changes are being considered on page 97, Tuesday, May 18th. Sac to Xba and HindIII to Xba, etc. Checking on the following pages. Pretty ghastly gel on page 100 with a comment despite the gel accident, etc., useful result. More site change minis page 103, 109. Confirmation of the site changes. [00:24:00] For example, the conclusion on page 108 that TK is correct. It now has an Xba site though it cuts poorly. Considering a control plasmid on pages 111 and 113.[00:25:00] Worrying about the difference in activity of a neo gene in two orientations on page 113 and 112. Revision of the construct on page 114. Experiments to get to where we wanted to. Looking on Thursday, June 3rd page 123 at the various fragments that were available [00:26:00] with regard to the TK gene and vector, etc.
Various possible constructs available with a 5’ untranslated region. TK gene and part neo gene. [00:27:00] Revise status June 17th, 1993. My sister Nancy’s birthday. Fragment from pMC1neo changing an Xho site to HindIII typical of what was needed in this sort of work and ending up with a conclusion that the change in site was 100% correct. [00:28:00] Back to tests of homologous versus non-homologous recombination now on page 135 Tuesday, June 8th TK versus ganciclovir with one, two, three different constructs being considered and the results being as usual. [00:29:00] No effect on the ratio of non-homologous to homologous recombination. I wonder that I went on doing this work so long. It was never successful. Looking at PCR primers for the ACE positive control, page 139. Trying out PCR for this on page 141. [00:30:00] So this was construct made so that it told us that the PCR reaction was working. Positive control plasmid which had the two primers corresponding to targeting already in the right position, and ending up with a conclusion on page 140 that number seven is the PCR control omega 110. Page 143 and 142 typical of the detailed [00:31:00] things that one had to do. Full of little notes. The whole two pages. On with the work. Tidying up gels, etc. Final test on HNTK and HNTK reverse, page 153, Wednesday, June 16th. The results which are on page 152 confirm no difference in HNTK and HNTK reverse in enrichment with ganciclovir. So negative result. [00:32:00] After all that work.
Coming to the end of the book. We’re ending on page 161, Sunday, June 20th. Reverse PCR control continued with a conclusion that one is in one orientation and one is in the other. Number 8, 35, and 36 are one orientation, 32 and 42 are the other, [00:33:00] and pick two to go on with 8 and 32. And so ends book omega. [00:33:11]