WEBVTT 00:00:06.680 --> 00:00:11.180 [Westphal, Heiner; Transgenic mammals and biotechnology (videorecording)] 00:00:11.180 --> 00:00:18.550 [This seminar presentation was originally videotaped for internal information purposes only and is made available now becuase of expressed interest in the subject material] 00:00:19.120 --> 00:00:24.090 [National Library of Medicine Biotechnology Seminar Series: An Update] 00:00:24.090 --> 00:00:37.030 [Transgenic Mammals and Biotechnology, Dr. Heiner Westphal, 16, Sept. 1988] 00:00:37.030 --> 00:00:59.980 Dr. Heiner Westphal: Thank you, Dr. [?], and thank you all for joining me this noon and discussing what becomes rather fashionable in biology, namely, to put, as the slide shows, genes that work in the living organism. 00:00:59.980 --> 00:01:17.410 This is no small feat for those of us who have been trying in the past to observe genes in the petri dish, where, for instance, a tumor virus might transform a cell. [Slide reading: The power of the transgenic technology lies inthe fact that genetic effects can be measured under real in vivo conditions at any state of development of the organism.] 00:01:17.410 --> 00:01:29.780 But if this virus transforms a tissue in a mouse, for instance, then we are less certain what really happens in this living organism. 00:01:29.780 --> 00:01:53.810 We can't reproduce the same event over and over again in order to decipher the biochemistry and genetics of the event, so what we really need is animals that have the same event occuring over and over again, and the same target cell, maybe at the same time of development. 00:01:53.810 --> 00:02:11.940 And such a system is possible by taking, for instance, to stay with our example, the gene out of the tumor virus and putting it in the germ line of mice so that it's now present in every cell of the body. 00:02:11.940 --> 00:02:25.690 And, depending on the controls that I exerted upon this gene, and I'm coming to those in a moment, the gene might be expressed in one or another organ in this mouse. 00:02:25.690 --> 00:02:39.980 And since it's in every cell, it's also in the germ cell, so the progeny of those mice will also express the gene, again, in specific target organs in specific times in development. 00:02:39.980 --> 00:02:48.610 And so we can do real biochemistry and genetics off transgene expression. 00:02:48.610 --> 00:03:01.970 Because this is what we call them, we call them genes coming in from the outside, and therefore, Prinster, who developed this technique early on in the 80s called them transgenes. 00:03:01.970 --> 00:03:32.430 Now, what is the technique? How do we bring a gene into a mouse? You see here that there is a piece of DNA, that you see here, indicated as a double helix, and it is entering a glass syringe and it makes its way through the syringe into here, which is the core nucleus of a one cell mouse embryo in this little sketch. 00:03:32.430 --> 00:03:51.430 And, if you're lucky, then, before that cell divides to make two and four and eight and the end, 10 to the 14, this gene will have integrated in the genome of the mouse, and so we have obtained a transgenic animal. 00:03:51.430 --> 00:04:10.540 Of course, we cannot keep this embryo by itself, it has to go in a mother. And so what we do is, we take the injected embryo and, well I can't see my little, here it is. 00:04:10.540 --> 00:04:28.330 We take it into the infundibulum, as it's called, of the oviduct, and it will go through the oviduct much as a fertilized embryo will do and finally wind up in the wall of the uterus after four days, 00:04:28.330 --> 00:04:49.680 and it needs another 17 days or so of gestation time before a little mouse is born, which now has all those transgenes, depending on what we injected in the various cells of the body. 00:04:49.680 --> 00:05:16.300 One of the transgenes that I wanted to show you, here, introduces us into the topic of today's talk, namely transgenic mammalian organisms and biotechnology. Biotechnology is a fast growing area of research where people are interested in finding ways of treating human diseases. 00:05:16.300 --> 00:05:33.390 This is usually the topic, of course, it can also be importing for agriculture, and certainly be important for plants. There are transgenic mice, there are transgenic plants. 00:05:33.390 --> 00:05:55.440 In this particular case, however, we want to limit the discussion to biotechnology as far as it pertains to the human disease, and one of the human diseases that our institution at the NIH puts much, much attention is cancer. 00:05:55.440 --> 00:06:12.120 So we have, here, constructed a gene which will cause cancer in the transgenic mouse. And the way we constructed this gene is by having a little promoter sequence, as we call it... 00:06:12.120 --> 00:06:36.550 this black box, here, which is located upstream, or, in this story, to the left of the actual gene. It contains elements that control this gene. And then this promoter is fused to a DNA sequence actually encoding for the gene that we want to express in the mouse. 00:06:36.550 --> 00:06:47.670 In this particular case, this is an oncogene, making an oncogene product, a tumor gene, with other words, that will be expressed somewhere in the mouse. 00:06:47.670 --> 00:07:02.310 It is this black box which is upstream that tells us where it will be expressed, because this black box is derived from a crystalline gene of the mouse, and the crystallines are the products of the lens in the eye. 00:07:02.310 --> 00:07:25.790 And so, with this construct, we direct the tumor gene product to be expressed starting at a certain time in the development of the lens. This temporal signal, which tells us when it will start to be expressed is also present in this little black box. 00:07:25.790 --> 00:07:40.590 And here you see in a adult mice, where the red dot is is the normal mouse and where it goes now is the transgenic mouse, you see that there is, indeed, a cataract, 00:07:40.590 --> 00:07:52.160 as we would call it, [?] in the lens of this animal that expresses the transgene and this is caused by a tumor that goes there. 00:07:52.160 --> 00:08:07.630 The aim of the experiment is not to show that this gene could produce a tumor, that was well-established even in the petri dish, many decades ago. 00:08:07.630 --> 00:08:38.040 But the aim of the experiment is to see whether we can learn something about differentiation in the normal organism and how it is perturbed by the tumor gene, and we hope, of course, once we understand that, we can have a little bit better handle on regimens of treatment of this disease. 00:08:38.040 --> 00:08:55.920 In this particular case, I told you the black box, the promoter of the cancer gene, sees to it that the gene is expressed very early in development. And here you see a pair of siblings, one is transgenic, the other one is not. 00:08:55.920 --> 00:09:08.900 This one is the normal sibling, and here, where the red dot is, is the [?], which is reddish, completely normal, but if you look at this little sibling here, he has a little white eye. 00:09:08.900 --> 00:09:33.790 So already, on day 15 of gestation, we see that a tumor is growing in the eye, so we know that we have to go very early if you want to know what is going on. Let's look at the lens development and see what changes in differentiation we expect in such a system. 00:09:33.790 --> 00:09:45.920 And here, for instance, is an early stage of a lens, where it is really a hollow ball of cells, these cells called the epithelia, here they are. 00:09:45.920 --> 00:10:04.190 And they secrete a capsule around them, which is right here, and about a few hours later in the life of the embryo, this hollow ball was on day ten/eleven, this one here is on day eleven/twelve of gestation. 00:10:04.190 --> 00:10:29.150 The epithelia here in the back of the lens have elongated, differentiated in the fibers, sort of pushed their nuclei ahead of them. And this differentiation is going on, finally, the lens is filled with fiber cells and down at the bottom you see a diagramatic picture of an adult lens. 00:10:29.150 --> 00:10:44.190 What we discuss in the next few minutes all happens in these two stages on top, in this stage and in that one. 00:10:44.190 --> 00:10:54.210 Now, I should tell you that while the lens differentiates into fiber, it starts producing the lens-specific proteins, the crystallines. 00:10:54.210 --> 00:11:11.510 And there are three families of them, the alpha family, which is this one here, and it starts very early on the eleven, and as the lens grows, the alpha crystalline sort of goes with every cell, every cell makes it. 00:11:11.510 --> 00:11:30.310 The beta crystallins start being made later and the gamma crystallins, here, are the latest to come. And so we have two good markers to go hand-in-hand with the development of the lens. 00:11:30.310 --> 00:11:52.020 One is morphology, namely the epithelia are out and the fibers are elongated and they show us about the differentiation. And the other one is to look at the crystallins, which, again, tell us about how differentiation proceeds. 00:11:52.020 --> 00:12:06.950 Now we have, and I think we might have to take the lights off, I think the video people don't like it, but I like it if you take the lights off, because you see better what's going on up there. 00:12:06.950 --> 00:12:25.810 And, well, let's hope they take them off, yeah. Here is one embryonic lens, here is another of a transgenic animal, and here is a third one yet of another transgenic animal. 00:12:25.810 --> 00:12:43.080 We have generated several lines of mice carrying this transgene and one of those lines has this appearance which is very much like the normal lens on day 11, where these fibers push in from the back. 00:12:43.080 --> 00:13:03.030 Except we see a few mitoses up front, here, and that is something that you won't see in a normal lens. So these cells sort of resume division, and that's the only thing that you can say, on that day of the embryo, which is different from normal. 00:13:03.030 --> 00:13:20.820 This one, here, has these mitosis all over the place and these cells are really not elongated fiber cells at all. Already, at this very early stage, they are rounded. And only a day later, the picture has very much changed. 00:13:20.820 --> 00:13:44.600 The normal lens has the normal fibers elongated here and filling the entire lumen, whereas this one, here, while it has also filled the lumen, has these cell mitoses, cell divisions all over the place, and, of course, this is much more pronounced in this line of mice. 00:13:44.600 --> 00:14:01.460 Again, I want to emphasize that we can look at every day in the development because we have all the progeny where one little mouse isn't exactly like its sibling because they integrated the tumor gene in their germination. 00:14:01.460 --> 00:14:25.490 Now, when we look at the tumor gene products, the SV-40 tumor antigens, in this particular case, then we see that the mouse that has the tumor very early has also these tumor antigens all over the place in the epithelia and also in the inside, in the cells that are are rounded and divide rapidly. 00:14:25.490 --> 00:14:46.430 This is taken on day 12 of embryonic life. Whereas the other mouse line, which has much more benign appearance at this stage, has the tumor antigen selectively in those elongating fiber cells. 00:14:46.430 --> 00:14:58.850 And this brings us to formulate a model which might explain what we are seeing here and tie it in with the development of the organ. 00:14:58.850 --> 00:15:16.940 Namely, we could postulate that this one mouse that we might call alpha T1 has the tumor antigens accumulating very early in the epithelia, where there is very little differentiation going on. 00:15:16.940 --> 00:15:36.850 Whereas, in the other mouse, they accumulate later, and only in those already-elongated fiber cells. At birth, we see a consequence of this difference in accumulation of tumor antigens. 00:15:36.850 --> 00:15:58.980 We see, up there, the normal lens. We see that, as we call them, the alpha T2 line, the one in which the tumor antigens began to accumulate in the elongated fibers, it still looks, pretty much, like a normal lens, although it's different, it still has those cell divisions going on. 00:15:58.980 --> 00:16:17.640 Whereas this, which we call the alpha T1 line, has now grown a full-fledged lens tumor which is now about to cross the capsule of the lens and go into the remaining tissues of the eye. 00:16:17.640 --> 00:16:32.970 And if you take these lenses out from these animals and transplant them under the skin of test animals, we'll see that the ones where the tumor was rather progressed, 00:16:32.970 --> 00:16:44.020 that they also, pretty rapidly, increase in diameter under the skin of new mice, that is to say, they are quite tumorigenic, these cells, these tumors. 00:16:44.020 --> 00:17:18.110 Whereas, from the other line, the tumors hardly double and we see may maybe a little bit of tumorigenicity in those lenses. Likewise, we can take cells out of those without lenses and establish them in the petri dish they will grow because the tumor antigens of SV-40 have immortalized them. 00:17:18.110 --> 00:17:47.080 And we can see that the alpha T1 line is a good example of such a line in these lanes, 1, 2, and 3, that they make hardly any of the crystallins which are characteristic of a differentiated cell whereas the alpha T2 line makes them all plentiful. 00:17:47.080 --> 00:17:59.400 So even in the adult lenses, there is a very strong reminder of the events that happened on day 11 and 12 of embryonic life. 00:17:59.400 --> 00:18:20.180 In the line that accumulated the key antigens early before differentiation could take place, there is no differentiation, no sign of it later on, where the other line, it was the other way around, the differentiation had taken place first and then the tumor antigens accumulated, there is indeed crystallins in this. 00:18:20.180 --> 00:18:39.550 By the way, on the side of the slide, which is all the way to the right, we see that the key antigens, up here the large T and down there the small T antigen, the oncogene products of SV-40 do accumulate in all those cell lines, 00:18:39.550 --> 00:18:51.180 that is to say, they are the motor of growth of these cells in the petri dish and certainly also in people. 00:18:51.180 --> 00:19:19.810 And so we come back to our model here and we can really find, now, plenty of evidence for the fact that, depending on when the oncogene products accumulate during development of a certain organ, we see either a very slow progression of cancer, as in this case, or a very fast progression of cancer, as in this case. 00:19:19.810 --> 00:19:42.170 And concurrently with that, these mice, here, die early because they have a rather ferociously-growing cancer, and these have a normal lifespan because, throughout their lives, these tumor cells remain rather benign, so to speak. 00:19:42.170 --> 00:20:05.560 And this, in turn, is quite important to know for human cancer, where similar things have been observed where certain tumors of people have rather differentiated cells in them, that is to say, "benign" and slow growing, 00:20:05.560 --> 00:20:31.530 and others have very literally terms of morphology or biochemistry that reminds you of normal cells of the body and those are usually the very "malignant" cancers that are so dangerous for the patient who has them. 00:20:31.530 --> 00:20:52.100 Now, in light of the time, I wanted to come to two different topics which also are, maybe a little bit more, in the realm of biotechnology and yet they also deal with human diseases. 00:20:52.100 --> 00:21:09.180 In this slide, we see two different transgene constructs that we inserted into our mice. Both of them have to do with the AIDS disease and with the virus that communicates this disease. 00:21:09.180 --> 00:21:27.070 What we had in mind is to generate, if possible, mouse models that would reflect certain properties of the human disease so that, by observing these mice, we could learn about the AIDS disease, and especially how to treat it. 00:21:27.070 --> 00:21:54.640 The upper of those two constructs has the promoter of the virogenome, the so-called "long term repeater," LTR, of the AIDS virus fused to a bacteria marker gene which is called CAT, here. 00:21:54.640 --> 00:22:20.180 And the lower of the two constructs has our good old friend, the black box, the crystallin promoter, fused to what we call a transcription activator of the AIDS virus, the TAT gene. 00:22:20.180 --> 00:22:35.250 Now, the purpose of putting these two gene constructs in the mouse was the following: the upper one, we hoped, would tell us where this viral promoter would be recognized in the body. 00:22:35.250 --> 00:23:00.370 Since every cell has it, we would be able to tell which cells would express it and which ones wouldn't...and the lower construct, I'll come back to that, would tell us whether we could actually activate the upper gene in a specific organ, namely in the lens. 00:23:00.370 --> 00:23:39.530 In this slide, we see that the LTR CAT gene, as exemplified by a little dot here, which shows that it is expressed, here for instance, that this CAT gene is active in quite a number of organ and tissue systems in our mice. In the thymus, in the eye, in the heart, in the spleen, and the tail. We have looked into that in some detail, for instance, here, using thymus sights. 00:23:39.530 --> 00:23:59.420 The cells of the thymus are the ones that are very much affected in AIDS disease, and so we took these cells out of our mice and checked whether they would make CAT activity, and sure enough, they do. You see it here, here for instance. 00:23:59.420 --> 00:24:17.520 But then we did an experiment to it which mimics something that happens to AIDS patients frequently in real life. We would infect them with human viruses, which, for instance, are often passed on among homosexuals, 00:24:17.520 --> 00:24:25.640 like cytomegalo virus or the other nerve viruses, and, lo and behold, this CAT signal shoots right through the roof. 00:24:25.640 --> 00:24:45.000 That is to say these other passenger virus of people that are going around activate this AIDS virus promoter dramatically, two hundred fold and more, in these mice. 00:24:45.000 --> 00:25:02.940 And therefore, we can learn from this model, right away, that it is very bad for AIDS patients, obviously, to be infected with cyotmegalo virus, especially during the latent phase, where the patient isn't a patient yet, 00:25:02.940 --> 00:25:24.940 where he is just zero positive for the AIDS virus, these viruses might come in and actually activate the AIDS virus so that the disease starts. And that might hold for cytomegalo, and for adeno and for many other viruses. 00:25:24.940 --> 00:25:39.160 Another important lesson we learned as we observed these LTR CAT mice, as we called them, the ones that have the promoter of the virus fused to the bacterial CAT gene, namely, 00:25:39.160 --> 00:25:57.200 that one of these cells that is most active in expressing these construct, a cell, with other words, that has factors that recognize the AIDS LTR, these cells are the so-called "Langerhans cells" of the skin. 00:25:57.200 --> 00:26:17.360 These cells have much more of this CAT activity than any other cell type that we could find in our mice. And, interestingly, these Langerhans cells are the very first to go from the skin of people that convert from zero positivity to the disease. 00:26:17.360 --> 00:26:35.130 Now, these Langerhans cells are microphages. They migrate into the skin from the bone marrow. They are related, of course, to other lymphocytes, but they are also quite different from, for instance the cells in the thymus or in the spleen. 00:26:35.130 --> 00:26:52.390 So this experiment, in itself, has been pivotal in directing attention to these Langerhans cells in the context of the AIDS disease. 00:26:52.390 --> 00:26:58.600 I promised you this experiment would show us invivo transactivation. 00:26:58.600 --> 00:27:24.810 One of the organs in which the CAT activity can be found is the lens of the eye, and we see, in our mice, that the fibers and the epithelia have all of this activity within the eye and so this distribution is very similar to that we find if we would, for instance, put the alpha crystallin in front of CAT. 00:27:24.810 --> 00:27:39.640 These mice, here, of course, have activity in many organs, because they have the bowel LTR fused to the CAT gene, but if you put the crystalline promoter in front of the CAT gene, then you get activity selectively in the eye. 00:27:39.640 --> 00:27:56.450 And so, with our two mice, one having this construct, the LTR, in front of the CAT gene, and the other one having the alpha crystalline promoter in front of the TAT transactivator, we would hope to see the following: 00:27:56.450 --> 00:28:07.910 we would see that, if we would mate those two, then progeny inheriting both transgenes would selectively transactivate the CAT activity in the eye. 00:28:07.910 --> 00:28:36.430 And that is because they would have the CAT activity only in the eye, the CAT activity in many organs, but the TAT would act on the LTR and increase the CAT signal. Let me show you the result of such a mating in this slide where a HIV LTR CAT animal had been mated with an alpha crystalline TAT animal. 00:28:36.430 --> 00:28:55.230 And we would get nine progeny and analyze them, all nine. And we would see, up here, the DNA signal which tells us that the LTR CAT construct is in, so it's number two, three, six, and seven of those nine progeny. 00:28:55.230 --> 00:29:19.170 And down here is the DAN signal that would tell us which of the progeny have the alpha crystalline TAT construct, number one, two, three, seven, and eight had it. And down here, the CAT activity has been measured in the lenses, in the eyes of these animals, as it were. 00:29:19.170 --> 00:29:35.400 And you see that there's one animal, here, which has only the CAT gene, but not the TAT gene, and that has a low signal of CAT. But this one, for instance, which has the CAT and the TAT gene, has dramatic increase in CAT activity in that lens. 00:29:35.400 --> 00:29:42.970 Do we really know that that is because the CAT activity modulated the transgene LTR CAT? 00:29:42.970 --> 00:29:59.360 Well, let's show the next slide, where we measured CAT activity in four different organs and we see that, in an animal which has only the CAT gene, these are the CAT activities that you measure. It doesn't matter what they mean, just look at the numbers. 00:29:59.360 --> 00:30:19.080 And up here, it's an animal which has both transgenes, and you see that it really has elevated, an elevated level of CAT activity in the eye, but the other three organs are exactly the same as in the column on the lefthand side. 00:30:19.080 --> 00:30:35.230 So you really see a very nice demonstration of an invivo transactivation brought about by two transgenes that were put in the same genome by this mating of the mice. 00:30:35.230 --> 00:31:11.230 Finally, I wanted to come to another example of biotechnology using our transgenic animals, and in this particular example, we doctored a gene in a very specific way so that it would allow us to produce a protein of therapeutic importance in the milk of our mice. 00:31:11.230 --> 00:31:36.120 Now, this experiment is important, not so much because we want to milk mice for human drugs, let's say, but because it shows an way of producing proteins in animals which could be very useful to produce them in large-scale for the treatment of people. 00:31:36.120 --> 00:31:51.560 The protein at stake, in this case, is the tissue plasminogen activator, a protein that's very useful to treat clotting disorders, and you all will have read about that in the papers. 00:31:51.560 --> 00:32:25.420 And this human TPA can be produced in, for instance, Chinese hamster cells growing in big fermentors which carry the human gene and express it in certain amounts. And it can be produced in certain viral systems, but a very nice way to produce it would be to produce this active protein, let's say, in the milk of cows. 00:32:25.420 --> 00:32:46.940 So if you had such a transgenic cow, and could milk it, and could produce this protein in gram amounts, from cow milk, you probably could fulfill the entire need of all people on this globe for TPA by millking just ten cows daily. 00:32:46.940 --> 00:32:55.260 And that would be very cheap because you wouldn't have to have big fermentors and companies who have them and run them. 00:32:55.260 --> 00:33:00.380 All you would have to have is a bail of hay, actually. [Laughter] 00:33:00.380 --> 00:33:24.330 So here you see the mammary-control DNA, it's a piece of a mammary gene that is part of the mouse whey acidic protein gene, a prominent protein that appears in milk. 00:33:24.330 --> 00:33:41.420 And this control area [?] that this gene is expressed in the mammary gland, and then that the protein made and shipped out to milk. And this promoter had been fused to the coding region for this TPA. 00:33:41.420 --> 00:33:52.650 So here is our hybrid gene, that went into the mouse embryo and mice were generated and tested for the presence of the transgenes, 00:33:52.650 --> 00:34:08.650 and when they were finally lactating, then we found, in their milk the TPA, which actually dissolved a fibrin clot in the petri dish. It was a very active enzyme. 00:34:08.650 --> 00:34:20.890 There was carrot in the milk of these mice, so these mice, they are over there in our lab, they are happily making a human protein of very important therapeutic value in their milk. 00:34:20.890 --> 00:34:30.110 And they're making quite a bit of it because these milk proteins, unlike any other proteins in fluid, are there in high concentrations. 00:34:30.110 --> 00:34:48.840 If you take, for instance, cells and grow them in a petri dish, you grow them let's say in a suspension culture, in a liter of medium with all the serum and so forth, then the total protein content of the liter of medium will be in terms of milligrams per liter. 00:34:48.840 --> 00:34:58.930 In milk, proteins are present in terms of grams per liter, so it's a fantastic concentration of protein that this liquid has. 00:34:58.930 --> 00:35:12.760 And, of course, it is a renewable source, it doesn't harm the animals to milk them, and they actually live and don't die, so it's actually a very good way of producing proteins of medical importance. 00:35:12.760 --> 00:35:34.760 Now let's look how this worked, and you see, here, the action of this TPA is actually to cleave plasminogen into plasmin, which is an active protease which then dissolves the fibrin clot. 00:35:34.760 --> 00:35:47.420 And here are our little mice, this is the transgenic animals, and, actually, the little ones are pretty happy, they don't mind eating a little bit of human TPA while they're growing up. 00:35:47.420 --> 00:36:10.710 And here you see this very acidic protein gene expressed in the epithelia that line the alveoli f the mammary gland. And here you see the very acidic protein army, the army that enclosed the protein in our mouse, 00:36:10.710 --> 00:36:27.950 increasing with time prior to lactation, the day of lactation is here, where it's very strong, and so our little transgenic army is also accumulating over time toward lactation. 00:36:27.950 --> 00:36:48.120 And here is, actually, the test, namely putting a drop of milk into a petri dish which has a hole in the middle and it's filled with fibrin, and you see how the milk is eating away on the fibrin. 00:36:48.120 --> 00:37:09.610 So I will stop here and just leave this as an example for you, how we can directly apply this technology to something useful in biotechnology and come to the very last slide, which looks a little bit into the future, namely the future of transgenic technology. 00:37:09.610 --> 00:37:31.700 Where in the future, it will be possible, and several labs are working on it, to introduce the transgene actually not into the mouse embryo, but rather in cells derived from it, the so-called embryonic stem cells, and in such a way that the DNA is not integrating at random somewhere in the genome, 00:37:31.700 --> 00:37:51.740 but in a very specific place, in a specific targeted gene. Then these cells can be selected, put in a syringe, and put back into a normal embryo, and a little chimeric mouse can be obtained, and from it, a mouse can be bred which has a mutation in this particular gene. 00:37:51.740 --> 00:38:12.750 And so, in the future, we hope to be able to produce, for instance, mouse mutants of very prevalent human genetic diseases such as the globulin diseases and industry would be the first to realize the potential of a mouse that mimics sickle cell anemia 00:38:12.750 --> 00:38:22.950 in order so that this mouse could be used as a model to test all sorts of regimens that might be useful for the patient. 00:38:22.950 --> 00:38:42.440 By the way, those of you who are interested in this latter aspect, which we call homologous gene targeting, are invited to come to the Masur auditorium on this coming Monday, where Dr. Mario Capecchi of Utah will give you the latest results of this type of technology. 00:38:42.440 --> 00:38:56.560 Well, I will finish my talk here and invite any questions that you might have, but before I do so, I wanted to acknowledge many people who have been helping these type of analyses, 00:38:56.560 --> 00:39:18.810 people who have done most in terms of analyzing tumors in the eyes of mice are Cathy Mahoney in my lab, and also Dr. Nakamura in my lab, Ken Nakamura, and we have done part of this work in collaboration with the laboratory of Dr. Joan Pendagowski (?). 00:39:18.810 --> 00:39:32.620 Then the work on HIV gene constructs has been done partly in collaboration with the lab of Michael Martin here at the NIH and also with that of Martin Rosenberg and Smith Kline French. 00:39:32.620 --> 00:39:46.070 And finally, the work with the milk proteins has been done in collaboration with the lab of Loder Heinichhausen here at the NIH, and with Integrated Genetics, a small company in Boston. 00:39:46.070 --> 00:39:53.280 Thank you. [Applause] 00:39:53.280 --> 00:39:56.100 Yes? 00:39:56.100 --> 00:40:14.900 Male Audience Member One: The microinjection seems such a, sort of, primitive thing. You alluded to that with the homologous gene replacement, but does it have a, sort of, predictable efficiency for any sort of DNA inserted? And how many copies go in the genome? And do they just randomly appear anywhere? 00:40:14.900 --> 00:40:35.300 Dr. Westphal: Unless we use selection procedures, indicated in this slide, the integration is certainly unpredictable, it may be totally random. How many copies we integrate is a little bit adjustable, sort of, in terms of how much DNA one places into the cell. 00:40:35.300 --> 00:40:55.440 Usually they are between one and ten copies that people nowadays integrate, and, very often, they appear at one site of integration and as a sort of bandwagon of head-to-tail links in the gene, indicating that, in the process of integrating, it gets replicated, somewhat. 00:40:55.440 --> 00:41:16.510 The frequency of this occurring depends very much on the DNA itself, how clean it is, too, the nature of it, the skill of the experimentor. But in our lab, for instance, Eric Lee, who does all of our integrations right now, has a success rate of one in five. 00:41:16.510 --> 00:41:28.730 That is to say, one in five embryos that he injects and transfers back into a foster mother will become a transgenic mouse. 00:41:35.540 --> 00:41:37.760 Yes? 00:41:37.760 --> 00:42:04.550 Male Audience Member Two: In the experiment where you're producing TPA in milk, that was fortuitous, I gather. Is there some way you can force them to be expressed in milk proteins? And how much TPA is circulating in those [?] Do they have any [?] deficiency or excessive number of [?] 00:42:04.550 --> 00:42:18.460 Dr. Westphal: It turns out that they're perfectly healthy, so as far as other cell systems or the blood, for instance, they seem to be all right and we have not actually gone to the trouble of trying to detect it in blood. 00:42:18.460 --> 00:42:34.720 But I would predict that it would be very little because the promoter is very specifically selecting the cell of the mammary gland. And from there, it goes out into milk by way of signal peptide structures that it has in the gene. 00:42:34.720 --> 00:42:51.690 That is to say, every protein is constructed such, on the genetic level, that it would be shipped out into the lumin of the alveoli and would make it into the milk, and would wind up there. 00:42:51.690 --> 00:42:58.870 However my chemical question is, of course, can it be easily purified from milk? Where will it appear, for instance? 00:42:58.870 --> 00:43:08.660 Well our TPA, we were lucky, appeared in the way, that is to say [?] of most of the casein in the whey, and then, of course, it's a cinch, I think, to purify. 00:43:08.660 --> 00:43:14.660 That, however, is, of course, a property of the protein of choice, and might differ from one to the other. 00:43:14.660 --> 00:43:28.890 It turns out, however, that most of these human proteins of therapeutic value, and that could also include, for instance, factor eight and nine as used by hemophiliacs, happen to be also found in small amounts in milk. 00:43:28.890 --> 00:43:41.660 So the body shows us that it's possible to actually have them in milk. Whether they can be purified from there will have to be seen. 00:43:45.770 --> 00:43:49.210 Yes? 00:43:49.210 --> 00:43:57.390 Female Audience Member One: Does the amount of TPA in the milk vary with the number of gene copies and according to stage of lactation? 00:43:57.390 --> 00:44:02.600 Dr. Westphal: The latter is certainly true. You see it in lactation, and not anywhere else. 00:44:02.600 --> 00:44:19.450 However, we have not accumulated enough experience with this particular transgene to see whether there is a direct correlation between certain indication sites, let's say, or numbers of copies, and the amount of TPA one gets. 00:44:19.450 --> 00:44:31.820 However, for many other data that people have with many other constructs in the transgenic technology, it's rather doubtful as to whether we would see more expression with more gene copies. 00:44:31.820 --> 00:44:40.440 It is probable that the [?] site from which the gene is more open, so to speak, and therefore better expressed. 00:44:40.440 --> 00:44:53.580 What we are working on right now is, rather, to make it a better gene to start with; giving it more elements in the control regions that would see to it that it expressed in high amounts. 00:44:53.580 --> 00:44:59.840 Female Audience Member One: And at the stage of lactation method, is there more TPA during early lactation than later on or... 00:44:59.840 --> 00:45:10.890 Dr. Westphal: We have not followed this very carefully, but I don't know if anyone is here from Dr. Heinichhausen's lab, they might be able to answer that question. 00:45:10.890 --> 00:45:16.600 Female Audience Member One: Thank you. 00:45:20.700 --> 00:45:21.500 Audience Member: Thank you very much. [Applause] 00:45:21.500 --> 00:45:27.170 Dr. Westphal: Thank you. 00:45:28.080 --> 00:47:29.390 [National Library of Medicine, Bethesda, MD 1988]