This is the beginning of Book C’, December 1st, 1994, running through July 3rd of 1995. It starts off with a reprint of, “Isolation of a cDNA encoding the vascular type — angiotensin II receptor.” So we can think about‑‑ type 1 angiotensin II receptor.
But the next work page is Thursday, December 1st, page 1, a revision of the angiotensin receptor 1A duplication experiment, making some different primers. And with the various [00:01:00] maps. Exon 1 probe — or ATR 1B, with a note that that should be 1A. Exon 1, 129 base-pair — Exon 1 probe, page 3, Monday, December 5th. (pause)
Here is some work with rat DNA, checking the rat Exon 1 fragment. Why I was wanting the rat is not very clear. [00:02:00] (pause) A primer design that might work also with the mouse, for the Exon 1 of the ATR 1A gene.
Still testing different ways of staining — or not staining — of recording stain gels, on Monday, January 10th. Video Tests [00:03:00] 13, with a zoom lens. Some checks, without any conclusions.
cDNA from T.J. Murphy, ATR cDNA, AT1, angiotensin I receptor cDNA that’s [00:04:00] obtained from T.J. Murphy. Being used to get a probe. (pause) Various fragments being tested, on Friday, January 20th, page 19 — and that the C’‑19 fragment is Exon 1. Here is a letter from T.J. Murphy, Emory School of Medicine, January 11th, 1995, with the rat vascular AT1 receptor cDNA, subcloned into [00:05:00] Bluescript. (pause) So T.J. Murphy was helping us by sending us a cDNA probe to help get things to work. Usual sorts of problems, trying to get a gene out.
A PNAS review and a grant renewal finished, page 27, Sunday, March 5th. More tests with the rat versus mouse, Thursday, March 9th, page [00:06:00] 33. (pause) Trying to get better probe. (pause)
The Ides of March, March 15th, Wednesday, page 45, trying to get a polymeric probe amplification. “Pretty good amplification of [00:07:00] the polymers,” is the conclusion. Trying to get a stronger probe by polymerizing it. And continuing with still to make probe. On Friday, March 17th, page 49, a new probe for the promoter region, about one kilobase from the promoter, thinking it might be a more specific sequence — on Kim and Nobuyo’s recommendation. (pause)[00:08:00] Looking at a new way of starting the PCR reaction, page 57, Tuesday, March twenty-third‑‑ using an antibody, monoclonal antibody, against the polymerase, and which would then be destroyed, after being heated. With a conclusion, though, that “The antibody start improved the yield slightly in all cases but didn’t improve the product.” So not terribly satisfactory. Tried again on page 59. “Only marginal effect.” (pause) [00:09:00] And better labeling conditions, Monday, March 27th, page 63. Labeling, page 65.
Now helping Naomichi, Naomichi Matsukawa, on page 67. He was looking for the 5’ end of the ANF receptor C gene, the clearance receptor gene. And helping him map his phages, on this page. (pause) Testing them on the following page, more [00:10:00] PCR tests, page 71. Not at all unusual — “Start again,” page 70.
More from T.J. Murphy, a plasmid ‑‑ six kilobases of 5’-flanking region of the rat AT1A receptor gene. And a look at his Figure 2, “Molecular Structure and Transcriptional Function of the Rat Vascular AT1A Angiotensin Receptor Gene” — from T.J. Murphy’s lab. [00:11:00] He was very helpful.
PCR1 tests, on — PCR checks of ATR 1B disruption. “Sylvia and Denise and Kimberly have a good chimera, at last,” on an ATR 1B-targeting construct. “They’re certain is, however, somewhat ambiguous, so test with PCR.” And the controls are good. “PCR controls are exactly correct. Repeat the samples with more diluted DNA.” Too concentrated. So retested, [00:12:00] on page 79, but disappointing. “Neither is positive, although Denise says that both case gave Southerns. But looking back at the gels,” Sylvia’s Southerns, “raises serious questions on whether 8‑D and 9‑B were partial digests rather correctly targeted.”
Back to basics, more PCR, Monday, May 15th, page 81. All are negative, except the control. Looking at possible false positives from targeting DNA, [00:13:00] on page 83. (pause) Looking at ATR 1B, final construct #8. And a map there of what was expected. [00:14:00] The homology on the 3’ and — homology and 5’ homology being clearly shown — with Nobuyo’s help. (pause)
Page 89, PCR on some “very slight-chance positives,” from Sylvia Hiller. “Conclusion: Negative but worth the try.” [00:15:00] Interpretation of the result, Wednesday, May 24th, page 93, giving maps of the targeting construct and the expected results with the positive control, and with the genomic DNA.
Checking on Sp1 digests, for the Southerns, of ATR 1B, on page 95, Thursday, May 25th. Usual sort of experiments.
Now, [00:16:00] “A week ago –” comments on page 97 — “a week ago in 756XP,” probably my Cessna 182, “en route to Teterboro, began to think of using phages of the injection variety to inject DNA into mammalian cells,” in other words, use the bacteriophage to inject DNA into a mammalian cell. But you would have to have a receptor for the phage. “LamB or, better, the Shigella variant could be used as a receptor in mammalian cell.” And began to have this idea of injecting DNA using a phage to inject DNA into mammalian cell, which now had a receptor [00:17:00] for the phage injection machinery. Specifically, my notes are, on page 96, Friday, May 19th, 1995, “I think that lambda infects E. cola by a maltose receptor. Need to understand current views as to the mechanism, pH, etc., for absorption. And therefore, transfer the maltose receptor, under a pMC1 promoter, into E14Tg2a cell, using G-folinate nonhomologous recombination, etc. Complicated scheme.[18:00] So this idea is being pursued, Tuesday, May 30th, page 101, design of the multi-foreign plasmid.
But still working with — plasmid Pnt-delta-tk.
Ok. So on Monday, June 5th, page 103, back to considering this old plasma, Pnt-delta-tk, which is a PGK promoter, driving Neo, with a poly(A) and then a PGK promoter driving the tk gene, and then also with a poly(A), trying to excise the second copy of the tk gene. Continuing that work, on [00:20:00] Tuesday, June 6th, page 105.
Going back to the lambda — this integration, the receptor side, the maltose receptor. “Next step for LamB,” page 107, Tuesday, June 6. (pause) So the next step, the plan is to put LamB into Pnt, cloned or [00:21:00] total PCR from E. Coli — so driving the LamB gene, the receptor, with a PGK promoter.
So speaking to Charles Roessner. Warned that the outer membrane channel may act as a water-leaking channel in mammalian cell, since the periplasmic space is not osmotically protected. In other words, be careful.
A strategy for cloning. This is discussed on page 109, Wednesday, June 7th. And various maps are [00:22:00] there. So more work on Pnt-delta-tk #26, with a map of what I’m expecting. Trying to get to the LamB — PCR from E. coli DNA, page 113, Saturday, June 10. [00:23:00] (pause) E. coli LamB PCR, page 117, Tuesday, June 13. “PCR is fine.” Continuing. First attempt at making the plasmid, page 121, Friday, June 16. “None have the insert.” [00:24:00] (pause) LamB clone from E. cola, streaked out, that was obtained from Dr. Hofning, a pastor in [Scituate?]. Nancy’s 65th birthday, Saturday, June 17th. An alternate rescue of PGK LamB. [00:25:00] It didn’t pass the ligation test this time. So the Shigella LamB gene obtained from Dr. Roessner arrived on Monday, June 19th, page 129. So working more on trying to get — And my brother’s 70th birthday, Friday, June 23rd, page 137, doing lots of minis, 72 minis — with several conclusions. “All except 7 [00:26:00] are deletion. Not unexpected,” etc. Thinking about the maltose induction, page 139.
And, page 143, Monday, June 26, looking at PGK LamB blue candidates. Don’t look good. Continuing on the next page, 145. And different E. coli DNA, using different E. coli, [00:27:00] recA-minus E. coli competent cell. “Transformations –” Tuesday, June 27th — “but no improvement. Redesign and start again.” And redesign of the linkers for cloning LamB, on Thursday, June 29, page 151. And continuing. Lambda B continued. Lambda B continued. Lambda B continued. All of these pages. And ending the book, on page 161, single-stranded lambda B PCR. No, not that, not single-stranded, Shigella, the Shigella LamB PCR, on [00:28:00] Monday, July 3rd, 1995, with proceed. And so the book ends, on page 163, that, the blue colonies, “All three are the same.” And the tests executed, etc. So still trying to get the correct clone for a Lam‑‑ a LamB attachment site for bacteriophages. And that ends Book C’. [00:28:56]