Oliver Smithies:[00:00:00] And now we’re on book pi. Begins March 17th, 1988 and goes through May 24th the same year. So quite a thick book for a short time. The beginning pages are just the diagrams of the three-temperature controller rewired to a new form and tested, which can be used either as a two-temperature or a three-temperature controller. If the anneal is turned to zero it behaves as a two-temperature controller. And circuit diagram is shown. And rather complicated diagrams on page four. [00:01:00] Thinking about working with ES cells again. Amplification of PCR with these cells. Problems with getting proper amplification with healthy ES cells that are Mycoplasma-free. Bev comments that the cells with Mycoplasma are sticky and suspects that the lysis is incomplete if the cells are normal. So some thoughts on how to get better lysis of the cell.
Thinking about colony PCR on Friday, June 3rd, page nine. Need to be able to detect on colony replicas the presence of [00:02:00] the recombinant fragment. Some thoughts on trying to do amplification of PCR on something hybridized already to an immobilization system. In other words in situ PCR which I know has since been used quite extensively in different circumstances.
Worrying a little about imprinting on Saturday, July 11th, [00:03:00] a topic that I’ve always found difficult to deal with. Going back to some old DNA work and its implication, Saturday, June 11th, 1988, page 13. Pat has analyzed Qiliang Li’s goat DNA sequence data upstream of epsilon, etc. Talks about this looking for a hypersensitive site. Just stop for a moment.
This business of amping up things [00:04:00] on colonies bound to something is continued on Monday, June 13th, page 15. Colony amping up. Two 1-square-centimeter nylon screens Zetabind were used to pick up DNA or to pick up cells and try to hybridize or to try to amplify this material. The results are none of the samples show anything. And even the primer is absorbed to the filters. So retest with nonspecific DNA to block the sites or try nitrocellulose. [00:05:00] But certainly nothing is happening easily.
Continue the idea on the next page. But also with and without carrier. No amplification visible on anything although at least the primers are now visible so one can see the primers are there all right. Of course these pieces of nitrocellulose were reduced in size to fit inside the PCR reaction vessel. [00:06:00] The gels are quite pretty showing everything is visible. But no amplification.
Still continuing this idea the following day. Still negative. An idea developed in conversation with Tom Petes on Tuesday, September 20th, 21st, page 21. Beta actin promoter going through poly-Gn through beta Gal where n is not divisible by three and [00:07:00] therefore the gene is inactive. The thought being that you could control gene. You could determine the number of Gs. Not clear. Let’s try that again. Tuesday, September 20th, 21st, page 21. Some ideas developed in conversation with Tom Petes regarding having a promoter separated from an indicator beta-galactosidase with a string of Gs and with an AUG methionine upstream of the Gs that one would be able to tell whether the number of Gs was divisible by three or not.[00:08:00] So a transgenic animal could be made with clonal sectors marked. Because it would be likely that the number of Gs would vary during development. So the animal would have various parts of the organism with the beta-galactosidase active or not.
So these ideas could be tested with [00:09:00] Nobuyo’s plasmid pSVHBgal. Which is – and there is a map of pSP64 or pSVHBgal which is pSP64 plasmid with a polyoma enhancer, TK promoter, leader sequence, from alcohol dehydrogenase. Dealdehyde maybe dehydro. ADH Leader. And a beta-galactosidase. With SV40 poly(A). So this is a TK beta-glactosidase plasmid [00:10:00] which she called HBgal. And with the idea of fusing this plasmid to put in a string of Gs. Replacing a portion of the ADH leader by inserting the G sequence.[00:11:00] Making modification of beta S major Wednesday, November 30th, page 27. [00:12:00] So resulting in the ligation, pi 26 ligation tested on page 29. Good ligation. And use for transformation. Two plasmids were obtained. Only two. The transformation was poor. So pi 28A and pi 28B which were tested on page 31, the following page. So on.
Minis were obtained from these two plasmids. [00:13:00] Digested with EcoRI and HindIII and conclusion that they repeat it with less sample. The gels were blown away. More thoughts on detecting homologous recombination Friday, February 10th, page 35. Brad’s experiment looking at the AB times CD and PCR amplification to find AD. Ran into problems. [00:14:00] Variable amplification and contamination.
And so to avoid the possibility of getting contamination try some other procedures. Some thought of using the steel locus or the c-kit, I forget which it is. The W locus. On page 37. [00:15:00] Use of W star dash plus heterozygotes. Because previous work from Evans had shown an increase in germline transmission when the recipient blastocyst W star W star and even Wv slash wild type. And clearly good results are to be expected when the host blastocyst is infertile but viable. Fertility is the only criterion, etc., etc. Thinking about this anyway. Whether it was worthwhile, not sure.[00:16:00] Think about a ligand for the W receptor which was going to be called W receptor for steel, WRESL. Talk to Stuart Bentley. Steel plus. And talking to Dana Fowlkes and Stuart Bentley and Jane Barker that steel plus is the same as W plus. Very hard to tell apart. Although steel steel is [00:17:00] lethal just before birth whereas WW homozygotes frequently die a little after birth. And so a hypothesis on how that might work.
And on the following page a paper on the steel steel D mouse bone marrow stroma, etc., etc. Talking about steel. A potential assay that could be worked out.
And [00:18:00] this whole idea of using things in relation to c-kit and steel required plasmids and so I was fortunate and Peter Besmer sent me a c-kit plasmid which I planned to use to determine the genotype of mice carrying the W mutation. A little letter from Peter. And the original shipment didn’t arrive and he sent a replacement.[00:19:00] Some more W locus mutant plasmids obtained from Dennis Stephenson in Verne Chapman’s lab. People would send DNA and samples very easily. It was always a delight. [00:20:00] More DNA. More samples from other people. Synthetic thymosin beta 4 for the initiation of our receptor studies, etc., etc. From Allan Goldstein at George Washington University.
So on with this thought. More DNA obtained P plan mid AK delta from Allan Bernstein. But now modified. Which is modified from Peter Besmer’s full-length clone, etc. [00:21:00] Aim to clone it to make a soluble product. A map. A gel photograph.[00:22:00] Trying to think about immortalizing cell. Wednesday, April 12th, page 53. Plasmid A small d5E1A is a fragment of the 12S RNA. It can be used to immortalize cells without transformation. With a multiplicity of 10 and very [00:23:00] little productive virus will be made, but nearly all the cells will be infected. What that was to be used for is not clear.
So still thinking about soluble wide spot receptor sol W to express the extracellular domain plus a FLAG peptide able to recognize thereby a monoclonal antibody. Use this for purification, etc., FLAG sequence. [00:24:00] And how to construct these various things sol W on page 58. There is a series of pages of mine. First time that I remember seeing a Bluescript being talked about. But a Bluescript SK could be used with there all the sites [00:25:00] in the EcoRI region. Terminated in HindIII and not one EcoRI site, etc. Beginning to start using Bluescript.[00:26:00] Still thinking about c-kit and various ways to handle that. [00:27:00] Can you by any chance let me hear that last bit? Or is it difficult? It’s always difficult. So here on page 60 in book pi [00:28:00] I’m talking about modifying PAK delta. PAK delta is the plasmid with a beta actin promoter driving c-kit that was described or shown on page 50. So it’s an expression vector for c-kit. Just a modification of sites being considered on page 60.
And Dan had suggested a modification. Modifying PAK delta, etc., etc. [00:29:00] And a couple pages of his thoughts there on page 62. Sylvia’s gels on PAK delta and PH beta APR1 cut, etc. With plasmid being talked about here PH beta APR1 is a map shown on page 66. It’s a fragment of the human beta actin gene. [00:30:00] Human beta actin promoter with a cap site. And a 5’ untranslated region. An IVS2 site and a cloning site. Which can be used to make expression vectors.
This is one of the earlier [00:31:00] references to Sylvia Hiller, who was the daughter of my first technician and great friend, her father. So Sylvia is running a gel here of digesting PAK delta upper and lower band, etc. Though the digest with BalI is very suspicious. [00:32:00] Next page of BalI digest of PAK delta, upper and lower band. Not as advertised. Prep gel for c-kit with Bal ends. Waiting for the enzyme.
Some oligos being purified [00:33:00] on Monday, May 1st. So one is now able to order these sequences. This is May 1st. So have now reached — I think this must — we must hope by now have reached North Carolina, because this looks like orders from the pathology department. Let me just see a little bit more about what’s happening at this stage.[00:34:00] So didn’t remember the exact date, but there is April 27th. Let’s see if we can work it out. [00:35:00] Because here is an oligonucleotide request for synthesizing an oligonucleotide with Dana Fowlkes’s help. He was in the department of pathology and I moved there and he was a great help in getting me settled down and Nobuyo of course. So here is an order for a linker now. One can order DNA. They had a DNA synthesizer, which was a great treat, to be able to order oligos instead of try to find them in some other way.
So this is the [00:36:00] beginning of work at North Carolina. Better enzyme than BalI. MscI being tested on PAK delta. Page 77. May 2nd. Linker ligations. Decided to run a prep gel anyway. Sample prepared with yeast tRNA carrier. [00:37:00] New procedure, prep gel. Continuing DNA work. Here we are. Page 85. Wednesday, May 4th, transforming Ron’s cells and protocol for using cell TH5 alpha F’. [00:38:00] Competent cells which can be bought from BRL. Rather than making competent cells one could buy them from BRL. Later on we regularly made our own. But still at this point just buying the cell TH5 alpha F1. Making the minis on Thursday, May 4th. With the conclusion on the preceding page, 86. [00:39:00] Number five being silicate is looking good. Let’s have Ian do more tests with it and proceed. So I don’t know whether I mentioned that Sylvia Hiller was the daughter of Otto Hiller, my long term friend, my first technician. But anyway going with pi 87 minis on May 4th, Thursday, working with number five, which we want to call PAK delta betaII. [00:40:00] Pi 86 number five going on checks on it and continuing to check it again on Sunday, May 7th.
So trying to insert new sites into the construct. DraI and HindIII fragment [00:41:00] going to be inserted into number five. Straightforward DNA work. Trying double-stranded oligos on page 105, Tuesday, May 9th, by [00:42:00] taking a synthetic oligo DraI-to-HindIII top strand and DraI-to-HindIII bottom strand, annealing them, and then using these. And the result, the conclusion is not satisfactory smear by UV and fluorescence. Not much better by ethidium bromide. The image is not there. DNA work of ordinary straightforward type. [00:43:00] Continuing DNA work. Monday, May 15th, page 117, etc. But this is pi 117 single cut HindIII. Nothing very remarkable. Just making a construct. Continuing page 121 and 123 and so on. [00:44:00] Straightforward DNA preparations. Alternate approach thought of on Wednesday, May 17th, page 129 and rejected. So Thursday, May 18th, just wait for bulk growth, etc. How to go ahead. More minis Sunday, May 21st, page 137. [00:45:00] Tested the minis on Monday, May 22nd. Several pages of tests. Repeating digests. Just making the DNA construct. So minis being checked on Tuesday, May 23rd, page 147. Various ones are now correct. Number 12, number 15, and number 18 now correct. Pick 18 for now. [00:46:00] So it continues to the end of this book, the last page being page — two scratched out pages. The last surviving page is page 159, Wednesday, May 24th, isolation of a HindIII EcoRI DraI [00:47:00] fragment 2.1-kb for use in further work. Pi 159 2.1 is the product. And that ends book pi. [00:47:19]