Oliver Smithies:

[00:00:00] This is book theta.  Beginning October 27th, 1983, and ending June, 1984.  So it’s a rather relatively long period in one book which we might find out why eventually.  But here we go.  Begins Thursday, October 27th.  Dideoxy and prep gel of Nar digest of pSV2neo delta left.  This is thinking about labeling or not labeling a digest dideoxy material.  [00:01:00] Can see why.  So this book begins on Thursday, October 27th page one.  And it’s following [00:02:00] up on the idea that was elaborated at the end of the previous book, which I think I’ll go back and just explain again.  On Saturday, October 22nd, the last pages of book eta describes how tests by Raju on my suggestion showed about a 30-fold increase in recombination of delta left neo and delta right neo plasmids in mammalian cells could be obtained by introducing a deletion caused by the enzyme Nar into delta left neo.  So there was a deletion in the plasmid that helps with — induce a repair mechanism.  And the idea then was to test the effects of [00:03:00] calf intestinal phosphatase on this reduced ligation and of dideoxy ends to prevent ligation.  So starting with Nar digest which are deletions of pSV2 delta left neo.  What would be the effect of different end treatments.  And that’s where book theta begins.  By putting dideoxy ends onto the Nar digest to prevent ligation.  So here we are.  Ligation test Friday, October 28th, page three, making the material to test this idea using Nar neo D dideoxy versus Nar neo.  [00:04:00] Just to see whether it does indeed prevent ligation.  And the conclusion is the yield of Nar dideoxy Nar neo dideoxy is rather low.  But ligation occurred in DD and in DD plus C, whatever the C was there.  Therefore the dideoxy didn’t work.  That was the conclusion.

I must have gone to a lecture by Paul Berg on Wednesday, the 2nd, because he was — there were some suggestions there that dihydrofolate reductase, [00:05:00] no, it’s not clear, I don’t really quite understand what that was.  But when dihydrofolate reductase is being used with a terminator, followed by a new methionine with neo that this is before two proteins from the same RNA were well understood.  But he did comment that this works if the neo gene — if the DHFR gene with the terminator and the neo gene with the methionine [00:06:00] work, if they’re not too far apart.  But if the termination is deleted, translation — if the stop codon, the terminator, is deleted, translation goes right through.  So TNFUS could be TN dot met FUS.  So maybe my incorrect 13 and 14 will work over the page idea.  Just different ways of getting around expressing two genes or using one promoter to express two genes.

So review of TNFUS 13 and 14 and of new TNFUS minis [00:07:00] on Thursday, November 3rd, page seven.  Continuing to work on the tk-neo fusion.  Back to 117 again on Tuesday, November 7th, page 19.  Delta beta 117 minis on a dam strain.  [00:08:00] So that this was beginning on a dam- strain.  With a comment that this BclI because unless one uses dam- strain we couldn’t use BclI.  The BclI site is needed for linearization outside the homology while BstXI site is needed for linearization inside the homology.  And this is growing the minis on the dam- strain [00:09:00] so that you don’t get modification of the BclI site to make it uncuttable.  Nar again on page 21.  Thursday, November 10th, page 23 is considering using or making a nicked circular pSV2neo delta left.  Since Nar nicks pSVneo delta left as well as cutting it, an easier control of nicked circles, etc.  [00:10:00] With the conclusion from the results that the DNA — that NarIII is only a trace better, etc., etc.  But the DNase is as good as I can make it.  A little supercoil left and a little single random cut, etc.  Help on blunt end ligations from a paper published in PNAS October 1983 from Zimmerman and Pheiffer that crowding with polyethylene glycol would help blunt end ligations by DNase ligase.  A purely technical problem but quite interesting anyway from a point of view of gel.  [00:11:00] So that is then added on page 24 and there is attempt at ligating with 15% PEG on the same page.  After filling in dideoxy.  So to get ligation even though the ends were blunt.  So here is theta 25 control and ligation and that’s K theta 25, K being Klenow material.  And DD theta 25 which is the dideoxy material.  And fairly good ligation of the control with 15% PEG.  [00:12:00] The dideoxy inhibits substantially but not completely about 70%.  So Raju is going to test mitomycin treatment and UV treatment of DNA.  And he’s already done calf intestinal alkaline phosphatase and ditto single-stranded-specific nicking.  Various ways of treating the DNA, which as I’ve already mentioned ended up as a paper from the two of us.  And he was evidently wanting to know what TNFUS number 31 was and I have an insert on page 28 that the map of TNT looked correct.  TNFUS number 31 still a bit suspicious.  [00:13:00] But that note to Raju.  This is the best description I can give of TNFUS 31 and TNT.  These are the designs and not proven structures.  But it shows what they were intended to be.  Sallie Boggs was a longtime friend and collaborator and came to the lab as a visiting professor.  And here is the first mention of her here.  A fairly complex insertion on page 33, [00:14:00] Wednesday, November 30th.  Suggested to Sallie Boggs and Raju Kucherlapati to use W mice from Sallie.  These are mice that are various mice which have a decrease in bone marrow competent cells.  Reduce erythropoiesis.  So take normal and thalassemic bone marrow.  Thalassemic bone marrow being from the W mice.  And calcium phosphate pSV2neo beta-globin gene DNA.  And select with G418 to get cells which have now got the neomycin resistance.  And then put back into the depleted mice or better use [00:15:00] into a WWv mouse.  That’s viable.  Wv is a viable W mutation.  Test for neo sequences, etc., etc.  Various — several combinations are possible.  In other words trying to think about correcting a thalassemic mouse with different materials.  And huge yellow sheet as a foldout there.  Showing various possibilities.  Too complex to try to resolve at this point.  But also a note.  Probably a lecture from Marshall Edgell.  November 28th, [00:16:00] 1983 talking about beta major, beta D major, and beta D minor, etc.  And maybe it’s a conversation with him, I think is more likely.  Getting these various constructs or various sequences of beta S major and beta S minor and the like.  [00:17:00] So various ideas on that page.  And looking at the Hbbd haplotype.  pSV2neo beta major construction now being considered on Wednesday, November 30th, page 35.  And maps of that region as an insert on the left.

So starting with Debbie Endean clones of the beta major beta D major sequence CA4 from Debbie Endean.  Minis on the beta major pSV2neo on Thursday, December 8th, page 37.  [00:18:00] Digestion no good.  Repeating again December 9th but none are correct yet.  More minis, etc., the following pages, remake of beta major slash neo Saturday, September 17th, page 45.  Beginning again.  Gel.  And calf intestinal phosphatase on the following pages.  And isolating fast, medium, and slow components from — on Monday, December 19th.  Conclusion, it’s a terrible gel, but proceed.  Expect tests to be reasonable but use it only [00:19:00] going on in that similar vein.  Transformation for this plasmid which is pSV2neo slash beta major which can be shortened as PN beta on Tuesday, December 20th, eight colonies were obtained.  And the conclusion that several of them are number 31 and four and five are correct orientation, number seven looks bigger, etc., etc.  Size is not quite correct so better do some more.

[00:20:00] Trying to nick with NarI, December 27th, Tuesday, page 55.  Didn’t work.  And then worrying about recombination at the phage level.  This is something that was a real concern.  But as we began to do this work we got more and more worried about the possibility that recombination really although we scored it correctly and found the recombinant fragment, the recombinant fragment had actually been generated during the assay.  That’s to say had been a phage phage or phage.  It required E. coli to get the recombination.  So it’s necessary [00:21:00] to find a design of a procedure which would allow one to test whether recombination occurred before or after the DNA had seen E. coli.  And this is the beginning of that thought though not the one that we finally used, I believe.  But it’s a retest of delta beta 17 by gamma beta HindIII fragment.  Test of the multiplicity of infection to exclude recombination at the phage level.  And if necessary even more dilute, etc.  [00:22:00] So that was the aim of this particular type of experiment.  So preparative gels on the next page will be made from number 31 theta 35 and number 4 30 53 to see the neo beta major and the neo.  Let’s try that one again.  [00:23:00] I see.  These are the two orientations.  Number 31.  Well, let’s start the other way around.  Number 4 theta 53 will be called neo major but 31 35 will be called neo jam, being major in the opposite orientation.  So there are maps of what one would expect from these two orientations on page 59.  Saturday, December 30th, both being about 11 kilobases long.  But having the different orientations of the beta globin major gene in this plasmid.  [00:24:00] SV40neo upstream and then beta major downstream.  And the other case with beta — with SV40neo and the reverse of beta major being downstream of the SV40 promoter.

Worrying about selection with G418 on page 61. Thursday, January 19th, problem most likely to give trouble with the neo beta major experiment is selection with G418.  Therefore set up a backup plan with the DHFR mutant as a dominant selectable marker instead of neo, wanted to make a dominant marker.  [00:25:00] And there’s a plasmid DHFR.  Plasmid which is described from a Simonson and Levinson plasma FR400 FR400, SV40 early phase promoter driving mutant DHFR which has low MTX affinity, etc.  And poly(A) site from hepatitis B virus.  [00:26:00] So this is an available plasmid.  The being in my case to cut it with Sal at a convenient Sal site and introduce a beta major gene on a 5.9-kilobase fragment into the SalI site by blunt end ligations.  And we now have on page 62 the map of the TNT 5 sequence and TNFUS 31 sequence available.  And showing that TNT is nearly correct.  It’s lost three bases but has a [00:27:00] terminator and initiator as planned and TNFUS.  That’s TNT is the terminator one.  And TNFUS is the fusion one.  TNFUS has a 50-base insert repeated twice.  And so really is a TNT.  That’s to say a terminator.  It could be corrected with XorII which is equivalent to PvuI.  So various ways out of that problem.  And TN 5 and TNFUS maps are then appended on page 65.

[00:28:00] Correcting TNFUS the 50-base-pair-too-long sequence due to the repeat on page 67.  Continuing on page 69 correcting the deletion.  Good ligation but the cells were dead.  So that page 69 didn’t work.  Here on page 70 is a DHFR mutant expression vector into pFR100, a map.  Presumably sent by the persons who sent me the original clones.  [00:29:00] Because of a design of a better beta major vector on page 71 using the mutant low MTX affinity DHFR gene as the selective agent in place of neo with a nine-step plan of action.  But I didn’t like the plan.  So a different plan on page 73.  Notes from Frank Burton on what might be done.  [00:30:00] So reclone, re-rescuing delta beta 17 or 117 for gene conversion.  Now beginning to think about using — about gene conversion rather than just insertions.  Delta beta 117 will assay plasmid integration but may be unable to assay gene conversion.  Giving an example there of what might happen by using delta beta 117 which eventually was the successful plasmid.  And so it’s recombination going to insertion.  OK.  But gene conversion would give something that would not end up as being useful, [00:31:00] because it would have lost the supF gene.  So I need homology across supF in order to see gene conversion.  With a note that all yeast work has agreed that gene conversion can skip large blocks of nonhomology but needs homology on the other side.  In other words gene conversion can occur without crossing over with a good diagram of it in the middle of page 75, delta beta with something like delta beta supF there and showing that it could really incorporate the sequence without ever having inserted the plasmid.  [00:32:00] And so a list of what to do on that bottom of the page.  Got signal with BstXI tail with Kpn prepare this.  Delta delta.  Tail with Kpn.  And clone and orient.  Etc., etc.  Looking at the sequences again to get out the 1.75 delta sequence and phage is available.  PPN101 at 275 micrograms per ml, etc.  [00:33:00] And Cla17 eta 119 is available at 173 micrograms per ml.  So the material is available.  And the experiment is then tried over the next few pages.  Single cut of Cla17 eta .23 protocol on page 81.  Kpn linker into the BS into the Cla hepska that was cutting with BstXI and then on the following page, Tuesday, March 27th, page 83, putting a Kpn linker into the Cla17 slash BstXI fragment.  With something from Raju Kucherlapati.  [00:34:00] Here are the data on the transfections of the genomic pSV2 delta left neo SV2 GBD and delta right plasmids, etc.  So things are going in that direction.

Proceeding with construction in the next few pages.  So making Kpn digest of Cla17 page 89.  The whole of Cla1 Kpn theta 87 now being treated with Cla intestinal alkaline phosphatase [00:35:00] to give us theta 89 Cla17 CIAP.  Ligation on page 91.  And diagnostic digest being considered on page 90 that if Cla17 gives an 8.0 and a 5.2 that isn’t right the desired is 9.8 plus 5.2.  Undesired 8.0 plus 7.0.  Etc.  Orientation can be checked later.  [00:36:00] Screened 32 minis.  Mike screened 32 minis.  Almost all colonies that were there.  And none were correct.  Too many were unmodified Cla17.  Therefore repeat the whole prep with prepurification of the delta beta piece.  Etc.  Just technical problems repeating the experiments needed to get the desired product.

Some more considerations on page 101, Saturday, May 5th with Raju, neo major and neo jam, the reverse results, [00:37:00] neo major gave 9.5 colonies per microgram and the reverse gave 6.25 colonies per microgram.  Some ideas on purifying circles and continuing to make the various fragments and ligations necessary to get the desired ends.  For example page 111 Friday, May 11th, ligation and transformation for [00:38:00] P delta delta dash delta beta only had trace ligation.  But left it longer and ligation was complete.  Proceed.  So the transformation on page 113 Sunday, May 13th, 15 colonies but it’s with a comment.  Hold these and repeat to get more.  So repeated to get more.  There are about 300 colonies on the next day and then hybridized them on Wednesday, May 16th, page 117 where a large number were positive with the hybridization to nick translated 1.8-kilobase delta beta piece from HBG1 a total of [00:39:00] 12 lists of which four were streaked for single colonies, A, B, C, D, 1, 2, 3, etc.

How to diagnose on page 119.  PstI can be used for the orientation and Kpn will tell the insert cuttability and HindIII is nice for simple length determination.  So start with HindIII.  With a conclusion that all but number 12 are Cla17.  Not delta beta delta beta.  Not delta delta delta beta.  [00:40:00] And number 12 is a deletion so all but 12 are Cla17 and number 12 is a deletion.  So pick 24 more and try again.  And the following page.  Minis for delta delta delta beta and number 33 may have an insert was the conclusion.  And so that mini was tested.  The experiment wasn’t convincing because I forgot to extract with phenol before trying to digest.

[00:41:00] Now comes a different thought here.  And I know what was in my mind although it’s not written in the book.  That was really due to a thought from my graduate student who pointed out that – a student, Karen Lyons pointed out that the experiments might never work if we went on using these bladder carcinoma cells because they didn’t express beta-globin at all and if we wanted to introduce a neo gene or whatever into that locus, A, we might not be able to get into the locus because [00:42:00] it was that part of the DNA was blocked up in heterochromatin or if we did get in there it wouldn’t be expressed because that region was not being expressed at all in these cells.  So it in a sense said your experiment will never work.  And the way out of this was therefore to try to use cells which already were known to express or be able to express beta-globin genes.  It turned out that there was one such cell line available and that was a mouse cell line that had a human chromosome 11 in it on which the beta-globin locus resides.  And that it was known to be inducible by various agents [00:43:00] to express the beta-globin genes and in other words this was a cell line that had the human locus in it and that the human locus was known to be expressible and therefore repeat the experiment in these cells instead of in the bladder carcinoma cells which Raju and I had been using all this time.  But then the problem arose that these cells don’t grow in a dish in the nice way that tk — that the bladder carcinoma cells grew.  But they were in suspension.  And therefore calcium phosphate could not be used to get transformants.  And so this was a problem that had to be tackled.  And [00:44:00] page 125 June 12th, Tuesday talks about the solution.

Well, Art Skoultchi had the right cells and he was a friend and collaborator and he had cells as I say with chromosome 11 in them.  But how to get the DNA into these cells.  But Hunt Potter had a paper that was in press which he had told me about, or sent, I don’t remember exactly how I knew.  But I talked to him or he talked to me.  In press in PNAS that you could get DNA into these cells which were in suspension by zapping them with a high voltage pulse, a DC pulse, which would punch holes in the cell membrane and allow DNA to get into the cell.  And if you had just the right level of punching as it were [00:45:00] you could get DNA into the cells without killing them all.  And so it was a balance between having enough holes in the cell to get the DNA in and having too many holes so that the cells died.  But he had produced a method of doing this using a particular type of power supply.  And I began to think about using this at this point.  So here on page 125, Tuesday, June 12th I’m talking about transformation of beta-globin-producing cell based on [00:46:00] the work of Hunt Potter which itself was based on preceding work by Neumann and others in EMBO Journal page 841 1982.  And Zimmermann and Vienken in Journal of Membrane Biology 67, etc.  Using high voltage pulses.  And here now so here we are.  ASF2 dash 1 cell from Art Skoultchi are MEL cells, mouse erythroleukemia cells.  They’re HPRT-.  They have 39 chromosomes which are normal but chromosome 40 is different and that chromosome 40 carries in it an [00:47:00] X11 translocation or carries on its end an X11 translocation which brings in the beta-globin locus.  And if HAT is used the chromosome X11 is retained.  Without keeping the selection there that chromosome is lost.  So it’s necessary to grow them under HAT selection or under HAT challenge all the time.  Making the hemoglobin, it’s at a low level.  But it can be induced with DMSO, which leads to differentiation and death, but nonetheless is inducible.  So these cells do make a little bit of hemoglobin [00:48:00] even without induction.  And certainly can be induced.  And so on.  Details of the cell that Hunt Potter used.  He had a cell with a 1-centimeter gap and using a 2,000-volt discharge from a particular power supply called ISCO 494.  And I looked up the circuit diagram of ISCO 494 and found out that it really had 200-microfarad capacitors bridged by a 47-kilobase resistor and there were six of these in series.  And so it was [00:49:00] something that I could make.  The circuit diagram is there on page 126, which in those days fortunately manufacturers would give you circuit diagrams.  And one can see what was involved.  The multiple copies of the series.  Copies of the condensers paralleled by resistors.  So I thought I could indeed make that if I wouldn’t have to buy one of these particular power supplies.

[00:50:00] So I made a power supply of that with that design.  I’m looking to see if I have the circuit diagram there, which I probably do, some state, because it was published.  Maybe not in the book.  But the idea then was to replicate the condenser resistor array of that power supply and use our high voltage power supply.  We already had high voltage power supplies.  And to load the capacitors up with the power supply.  And then trigger their release, which was [00:51:00] going to be done with a transistor or a slightly different type of control diode.  What we’re doing or what page I was on by any chance.  Can we find that?  Maybe picking up again on page 127.  The idea being to replicate the construct or the zapping that Hunt Potter had devised.  And trying to do this with 2,000 volts [00:52:00] from charging a capacitor to 2,000 volts.  But the difficulty was as we’ll see that a lot of the energy disappears into the spark when one does that simple experiment of charging a capacitor and then discharging it through the cell containing the cells that one wishes to transform.  So trying this at first and no signs of lysis at 2,000 volts.  And so pursuing this thought on page 129, Wednesday, June 13th.  The circuit diagram of ISCO 494 [00:53:00] suggests that the effective capacity is only 20 microfarads.  And the external resistance is about 200 ohms to the cells.  So that the time constant is approximately 4 milliseconds.  Whereas Zimmermann et al talk about time constants closer to 5 microseconds with capacitors of about 20 nanofarads, not 20 microfarads.  So I constructed a cell with a gap of 2 millimeters to permit operation at 10,000 volts per centimeter, the total voltage which I had available, 2,000 volts or less than that.  Where the time constant would be [00:54:00] about 20 microseconds.  And for this the capacity could be calculated and the resistance.  So here was the design of the cell, 10 millimeters cross section in one direction, 4 millimeters high, and 2 millimeters gap.  And the resistance should be 42 ohms, etc.  And if the capacity was 100 nanofarads, which I had a high voltage capacitor available, then I might be able to get it to work.  So I had a circuit.  I made a circuit on page 133 with a power supply charging through a 1-megohm resistor because there was no need to have a very rapid charge.  And measuring the voltage and having a switch which could be used to make contact.  [00:55:00] And found that this could handle — this simple circuit could handle anything between 0 and 3,000 volts with a zap frequency of about less than or equal to 1 per second.  And so I could use 3 zaps in 6 seconds and did this type of experiment.  So with an error.  I forgot to add the distilled water to the solution.

Continuing this general thought on [00:56:00] the following pages with very good comment from my old collaborator and friend Les Findrich in Toronto who was in my mind an electronic genius.  And he suggested that the spark would consume an appreciable part of the energy.  And that the actual delta voltage across the test cell is lower than expected.  So you have to do it by looking to see what happens with a scope by looking at the — with a Tektronix scope and so was a test made of this general type with a comment that this was tested with this scope.  The loss in the spark at 2,000 volts was not measurable.  Meaning that I wasn’t able to do it well enough.

[00:57:00] Anyway, going on with the zapping and survival I found that at 1 kilovolt or at 500 volts no cells were killed.  At 1 kilovolt about two-thirds of the cells were dead.  And at 2 kilovolts basically 100% killed.  So you could find something in between.  Beginning to do that type of experiment.  Three pulses at about 3-second spacing.  Room temperature with a voltage across the capacitor being 0, 500, 1,000, 2,000, 3,000 volts on page 137, Tuesday, April 19th.  The conclusion that the data [00:58:00] showed very steep kill curve in agreement with Neumann et al.

But let’s continue to go on with DNA transfer by zapping.  Wednesday, June 20th, page 139.  Attempt to transfer at room temperature, 4 times 10 to the 7 cells available in 40 ml, got them back up into a small volume, about 50% survival at various voltages, 800 volts, 3 zaps, 900 volts, 1,000 volts, 700 volts.  Different ways of trying it.  And using [00:59:00] dye exclusion to estimate how many cells were alive and how many dead.  One could get some idea as to what was happening in the different procedures.  So that about 60% excluded the dye at 700 volts.  And already easier to see the number that were dead, 91% were killed at 900 volts, 84% were killed at 1,000 volts, 14% were killed at 800 volts, and 14% and 40 at 700 volts.  Probably have those a bit wrong but one can see what the experiment was.

[01:00:00] Cell counts on Thursday showed that — better list a bit, 17% were dead with protocol one, up to 34% dead with protocol number four.  So the counts are not precise but at least it’s working.  So this type of experiment was now continued by DNA by zapping.  With an interlude to try delta delta delta beta 117 again again.  Friday, June 22nd, page 147.  So that type of experiment being talked about on Monday, June 25th.  Back to transformation again.  [01:01:00] On June 26th.  And back to cell zapping on page 153, Tuesday, June 26th.  I made a new zapper where there were one, two, three, four, six, seven capacitors in series.  Each bridged with a 1-megohm resistor so that the voltage would be spread uniformly across the capacitors.  And then charged up with a power supply.  [01:02:00] The time constant should be slow.  And set up then.  The capacitors were 100-microfarad.  But when in series this is equivalent to a 14-microfarad total capacitor.  And I found that I could get 3,600 volts.  The capacitor working limit is 3,200.  Etc.  And cell survivals with this type of procedure.

And more zapping on Wednesday, June 27th, page 155, 1.9 times 10 to the 7 cells available.  Zapped them at 3,600 volts, the maximum available.  [01:03:00] Zapped twice at 10-second interval.  And still no killing.  So consider going to the medium.  Also remember Neumann et al used trypsinized cells.  Back to minis again for delta delta on page 157.  Large number of positives.  But the experiment was not considered satisfactory.

So the book ends with a fresh look at delta delta delta beta.   Indications are that this Kpn linker is not working, etc.  [01:04:00] Start again with a new linker.  And so that ends book theta.  Just want to look at this and see whether — what happens here, whether — [01:04:21]