1 00:00:15 --> 00:00:20 We're going to discuss today the definition that you two gave which 2 00:00:20 --> 00:00:25 is making a genetically identical copy in terms of organisms. 3 00:00:25 --> 00:00:30 And I want to begin by distinguishing with you the terms 4 00:00:30 --> 00:00:35 reproductive and therapeutic cloning. 5 00:00:35 --> 00:00:44 Reproductive cloning refers to 6 00:00:44 --> 00:00:48 making a whole organism, doing cloning with the intent, 7 00:00:48 --> 00:00:53 and here we are in our board of life, same place we were last time, 8 00:00:53 --> 00:00:58 stem cells cloning, let's not dwell there. Reproductive cloning, 9 00:00:58 --> 00:01:03 the stuff that covers' of Time Magazine are made of. 10 00:01:03 --> 00:01:07 The outcomes of reproductive cloning, the desired outcome is that you get 11 00:01:07 --> 00:01:15 the whole organism. 12 00:01:15 --> 00:01:21 And I want to distinguish that from therapeutic cloning where the 13 00:01:21 --> 00:01:28 desired outcome is that you get some kind of cells and specifically 14 00:01:28 --> 00:01:34 stem cells. So reproductive cloning is the stuff 15 00:01:34 --> 00:01:39 of movies, it's the stuff of science fiction and of people having fun 16 00:01:39 --> 00:01:44 with covers of the New York Times Magazine. It's also the subject of 17 00:01:44 --> 00:01:49 scams. And I'll tell you at the outset, there are no human clones 18 00:01:49 --> 00:01:54 now. Human cloning has not yet worked, but I am sure someone 19 00:01:54 --> 00:01:59 somewhere is trying. And we'll come back to that later 20 00:01:59 --> 00:02:04 one in the lecture. The other type of cloning, 21 00:02:04 --> 00:02:09 therapeutic cloning is something that extends directly from our 22 00:02:09 --> 00:02:14 lecture last time. When you think about stem cells and 23 00:02:14 --> 00:02:19 you think about using those in repair, one of the issues that comes 24 00:02:19 --> 00:02:25 up, and this comes up in any kind of repair mechanism, 25 00:02:25 --> 00:02:30 repair therapy, transplantation of any kind is the question of autology 26 00:02:30 --> 00:02:35 or self or matching, whether or not the donated tissue 27 00:02:35 --> 00:02:40 genetically matches your own If it doesn't your immune system is 28 00:02:40 --> 00:02:44 going to try to reject it, and if it does you have a lot of 29 00:02:44 --> 00:02:48 problems. The sense of therapeutic cloning is that one may be able to 30 00:02:48 --> 00:02:52 take cells from an adult person and turn them into an embryo and then 31 00:02:52 --> 00:02:56 use the embryo cells, I'll tell you how in a moment, 32 00:02:56 --> 00:03:00 and then use the resulting embryo and the inner cell mass of the 33 00:03:00 --> 00:03:04 embryo to make various stem cell lines, as we discussed 34 00:03:04 --> 00:03:08 in last lecture. And the promise of therapeutic 35 00:03:08 --> 00:03:13 cloning is this autologous nature to it, that you would get something 36 00:03:13 --> 00:03:18 that was an exact tissue match. So let's discuss a bit more, why 37 00:03:18 --> 00:03:22 one might want to do this various types of cloning. 38 00:03:22 --> 00:03:27 In reproductive cloning there are some senses of replacing people who 39 00:03:27 --> 00:03:32 have been lost, some way of improving or overcoming 40 00:03:32 --> 00:03:37 infertility. Spare parts, this is the stuff that 41 00:03:37 --> 00:03:41 some good books have been made of. House of the Scorpion from Nancy 42 00:03:41 --> 00:03:45 Farmer. If any of you have read that it's directly relevant to this. 43 00:03:45 --> 00:03:49 If you haven't, House of the Scorpion it's called. 44 00:03:49 --> 00:03:53 It's a very interesting book that thinks about a time in our society 45 00:03:53 --> 00:03:57 where we can clone exact copies of ourselves. And these copies are 46 00:03:57 --> 00:04:02 made brain damages. And they are kept in hospitals and 47 00:04:02 --> 00:04:06 they are used for spare parts as the real person ages. 48 00:04:06 --> 00:04:11 OK? Sounds weird, sounds peculiar, sounds unethical, 49 00:04:11 --> 00:04:15 but it is possible. And I suspect there are people who would be quite 50 00:04:15 --> 00:04:20 keen on having this as something that could be done. 51 00:04:20 --> 00:04:24 Useful producer animals, there's been a sense in the 52 00:04:24 --> 00:04:29 agricultural industry that if one can make identical copies of 53 00:04:29 --> 00:04:33 particular animals it would be very useful in getting whole herds of 54 00:04:33 --> 00:04:38 goats, for example, that are making very high quantities 55 00:04:38 --> 00:04:42 of a protein that is pharmaceutically useful, 56 00:04:42 --> 00:04:47 some kind of drug for example. And there are in different ways. 57 00:04:47 --> 00:04:51 That kind of approach has already taken. Therapeutic cloning, 58 00:04:51 --> 00:04:56 as I said, autologous stem cells and the use, which I may or may not have 59 00:04:56 --> 00:05:00 time to touch on, of these cells in correcting genes 60 00:05:00 --> 00:05:06 which are mutant and which are bad. But let's step back for a moment 61 00:05:06 --> 00:05:12 because the question of cloning has a long history. 62 00:05:12 --> 00:05:18 And its history really started with a question that we have dwelt on for 63 00:05:18 --> 00:05:24 a number of lectures, and this is the question of how cell 64 00:05:24 --> 00:05:30 types are determined. And the question of when a cell type 65 00:05:30 --> 00:05:36 is determined, whether or not that is accompanied 66 00:05:36 --> 00:05:42 by some irrevocable change in the DNA. So this cell type 67 00:05:42 --> 00:05:49 determination, or let's say differentiation because 68 00:05:49 --> 00:05:55 that's the end point, as you know, is cell type 69 00:05:55 --> 00:06:02 differentiation caused by DNA change? 70 00:06:02 --> 00:06:06 Or let me actually say DNA change or DNA loss, which is the big one. 71 00:06:06 --> 00:06:11 And this question, and you could imagine, as you form a retinal cell 72 00:06:11 --> 00:06:15 or as you form a skin cell, you might get these different cell 73 00:06:15 --> 00:06:20 types because you throw out, you actually delete from the genome 74 00:06:20 --> 00:06:25 whole batteries of genes. OK? Or you make lots of copies of 75 00:06:25 --> 00:06:30 another gene. So you may actually change the DNA. 76 00:06:30 --> 00:06:33 You kind of know the answer to this question because I've already told 77 00:06:33 --> 00:06:37 you that all cells have pretty much got the same amount of DNA, 78 00:06:37 --> 00:06:41 the same type of DNA. And what's important is whether or not 79 00:06:41 --> 00:06:44 different genes are active or not. But how did I tell you that? How 80 00:06:44 --> 00:06:48 was I able to tell you that? So let's spend a few minutes 81 00:06:48 --> 00:06:52 exploring that. So the first question that was 82 00:06:52 --> 00:06:56 asked in terms of the cell type differentiation question was the 83 00:06:56 --> 00:07:06 question of cellular potency. 84 00:07:06 --> 00:07:10 Where cellular potency, and we're on number two of your 85 00:07:10 --> 00:07:14 handout, where cellular potency asked in an early embryo, 86 00:07:14 --> 00:07:19 when can an early embryonic cell or set of cells support growth of the 87 00:07:19 --> 00:07:23 entire organism? In other words, at what time during 88 00:07:23 --> 00:07:27 development is there totipotency, is there complete information in the 89 00:07:27 --> 00:07:32 cell? And this is to exemplify the kind of 90 00:07:32 --> 00:07:36 experiment that was done, as exemplified here in the sea 91 00:07:36 --> 00:07:40 urchin where you can take an eight cell stage sea urchin embryo and 92 00:07:40 --> 00:07:45 transect it in one plane or another plane. And if you do it in one 93 00:07:45 --> 00:07:49 plane you get an abnormal embryo and a normal embryo. 94 00:07:49 --> 00:07:53 They say abnormal but it's sort of normal-ish. And in the other plane 95 00:07:53 --> 00:07:58 you get out two normal, but small, larvae. OK? 96 00:07:58 --> 00:08:02 And what this said was that the cells of this eight cell embryo are 97 00:08:02 --> 00:08:07 not equivalent to one another. And, indeed, even four cells put 98 00:08:07 --> 00:08:11 together, if you bisect in the wrong plane, was not sufficient to confer 99 00:08:11 --> 00:08:16 totipotency to those cells. So at the eight cell stage in the 100 00:08:16 --> 00:08:21 sea urchin, the cells are not totipotent. And you can do similar 101 00:08:21 --> 00:08:26 types of experiments in many organisms. 102 00:08:26 --> 00:08:30 And what you find is that in the very early embryo, 103 00:08:30 --> 00:08:34 you can often split the embryo into its component cells and you can get 104 00:08:34 --> 00:08:39 out a number of genetically identical organisms. 105 00:08:39 --> 00:08:43 This clearly happens in humans in the case of identical twins. 106 00:08:43 --> 00:08:47 It happens in armadillos routinely where armadillos, 107 00:08:47 --> 00:08:52 for their own reasons, split into eight cells at the eight 108 00:08:52 --> 00:08:56 cell stage, and you get identical octuplets out of most armadillo 109 00:08:56 --> 00:09:00 pregnancies. OK? So there's totipotency but it's 110 00:09:00 --> 00:09:05 transient. And this transient nature of this 111 00:09:05 --> 00:09:10 cellular potency, so the embryonic potency is 112 00:09:10 --> 00:09:18 transient. 113 00:09:18 --> 00:09:24 And the question was then raised why? Was it something that happened to 114 00:09:24 --> 00:09:30 the cytoplasm or was it something that happened to the nucleus? 115 00:09:30 --> 00:09:33 And this is where we get into the questions of nuclear potency and the 116 00:09:33 --> 00:09:37 beginnings of cloning as we know it. 117 00:09:37 --> 00:09:44 And the technique that you need to 118 00:09:44 --> 00:09:49 know is called somatic cell nuclear transfer. 119 00:09:49 --> 00:09:59 Abbreviated SCNT. 120 00:09:59 --> 00:10:04 All right. So this is what SCNT looks like. This is number three on 121 00:10:04 --> 00:10:09 your handouts. And it goes like this. 122 00:10:09 --> 00:10:14 One takes an egg, early embryonic cell that you know is able to 123 00:10:14 --> 00:10:19 support total embryonic development with its own nucleus, 124 00:10:19 --> 00:10:24 and you go in and you remove the egg chromosomes. You can do this 125 00:10:24 --> 00:10:30 because most eggs are arrested at meiotic metaphase two. 126 00:10:30 --> 00:10:34 The chromosomes are condensed and they are gelatinous, 127 00:10:34 --> 00:10:38 and you can actually remove them. I'll show you a movie of this in a 128 00:10:38 --> 00:10:42 moment. You can remove these egg chromosomes, and you can then get a 129 00:10:42 --> 00:10:46 nucleus from some adult or other cell type. And you can put it in a 130 00:10:46 --> 00:10:50 glass pipette, and you can pipette it into the 131 00:10:50 --> 00:10:54 enucleated egg. The egg heals up. 132 00:10:54 --> 00:10:58 It's now got a substituted nucleus. And you can go and you can ask what 133 00:10:58 --> 00:11:03 this egg which its somatic nucleus can do. 134 00:11:03 --> 00:11:07 And the somatic cell nucleus is diploid so you don't need 135 00:11:07 --> 00:11:11 fertilization because you've already got the right complement of 136 00:11:11 --> 00:11:16 chromosomes. And if you do that, as was done in frogs in the early 137 00:11:16 --> 00:11:20 1960s, you get results like this. The host egg came from a frog which 138 00:11:20 --> 00:11:25 was brown and the donor nucleus came from a frog, or cells of a frog 139 00:11:25 --> 00:11:29 which was albino. And that was important because when 140 00:11:29 --> 00:11:34 you looked at all the cloned frogs they were all albinos. 141 00:11:34 --> 00:11:38 OK. And so you could see that they came from the donor nuclei. 142 00:11:38 --> 00:11:43 Great question. 143 00:11:43 --> 00:11:47 See me afterwards. I actually don't know the answer. 144 00:11:47 --> 00:11:50 That's a great question. OK. Here are the cloned frogs. 145 00:11:50 --> 00:11:54 OK? And you can get buckets full of these things, 146 00:11:54 --> 00:11:58 but. I've seen buckets full of these things. OK. 147 00:11:58 --> 00:12:02 But. OK. But before the but. So let's do a couple of things here. 148 00:12:02 --> 00:12:06 So want I can tell you is that in a 500 cell frog embryo, 149 00:12:06 --> 00:12:11 one can very clearly distinguish cellular potency and nuclear potency 150 00:12:11 --> 00:12:15 differences. So if you take any cells from a 500 cell frog embryo, 151 00:12:15 --> 00:12:20 you isolate the single cells and culture them as single cells, 152 00:12:20 --> 00:12:24 none of them will form an embryo. However, if you isolate nuclei from 153 00:12:24 --> 00:12:29 each of those cells and transplant those nuclei each into an enucleated 154 00:12:29 --> 00:12:34 egg, 100% of them will form normal embryos. 155 00:12:34 --> 00:12:38 So there is a massive difference between cellular and nuclear potency 156 00:12:38 --> 00:12:43 at that stage. It tells you that the nuclei are 157 00:12:43 --> 00:12:48 totipotent and the problem was with the cytoplasm of the cell and that 158 00:12:48 --> 00:12:52 the egg cytoplasm has got something in it that is special to support 159 00:12:52 --> 00:12:57 development. So that was a very positive result, 160 00:12:57 --> 00:13:02 OK, that you could get these cloned embryos, these cloned frogs from 161 00:13:02 --> 00:13:07 nuclei that by cellular experiments were not totipotent anymore, but -- 162 00:13:07 --> 00:13:11 But, and this is really important, if you did that experiment with 163 00:13:11 --> 00:13:16 nuclei derived from older and older embryo, here's blastula, 164 00:13:16 --> 00:13:20 gastrula, neurula tail bud and so on all the way up into adult cells, 165 00:13:20 --> 00:13:25 the frequency of embryos that arose from, or frogs that arose from the 166 00:13:25 --> 00:13:30 somatic cell nuclear transfer plummeted. 167 00:13:30 --> 00:13:33 So if you did the experiment at blastula stages you got 80%, 168 00:13:33 --> 00:13:37 even 100% success rate with the cloning with your somatic cell 169 00:13:37 --> 00:13:41 nuclear transfer. If you did it at gastrula it would 170 00:13:41 --> 00:13:45 drop to 50%. By late gastrula, which is just in frogs, about five 171 00:13:45 --> 00:13:49 hours after late blastula, you were down at around 15%. 172 00:13:49 --> 00:13:53 And by the time a day later had gone by and you were a tail bud 173 00:13:53 --> 00:13:57 embryo, you were close to zero percent success. 174 00:13:57 --> 00:14:01 So although nuclei were totipotent at some stage, 175 00:14:01 --> 00:14:05 there was a tremendous decrease in the potency of the nuclei as 176 00:14:05 --> 00:14:09 development went on. And so that raised issues, 177 00:14:09 --> 00:14:13 which we'll come back to in a little bit. Now, after the success with 178 00:14:13 --> 00:14:18 frogs, people started looking to mammals immediately to see if they 179 00:14:18 --> 00:14:22 could clone mammals. And there is a real checkered 180 00:14:22 --> 00:14:26 history here and some shady science and some claims particularly that in 181 00:14:26 --> 00:14:30 mice one could clone mouse embryos and could do it with 182 00:14:30 --> 00:14:35 high efficiency. And it turned out that all of that 183 00:14:35 --> 00:14:40 data was faked, and that really put a damper on the 184 00:14:40 --> 00:14:46 field for several decades. And it wasn't until the folks in 185 00:14:46 --> 00:14:51 Edinburgh in 1997 tried again and did it with an open mind that they 186 00:14:51 --> 00:14:56 were able to clone this sheep, Dolly, who never knew how famous she 187 00:14:56 --> 00:15:02 was, who was the first mammal who was cloned by nuclear transfer. 188 00:15:02 --> 00:15:05 So how does nuclear transfer work in mammalian embryos? 189 00:15:05 --> 00:15:09 The principle is identical to what I've shown you in the frogs, 190 00:15:09 --> 00:15:12 but I'm going to show you a few movies to give you a sense of how 191 00:15:12 --> 00:15:16 it's really done. So this is a microscope that's set 192 00:15:16 --> 00:15:19 up for somatic cell nuclear transfer. And these things here are 193 00:15:19 --> 00:15:23 micromanipulators, including injection and suction 194 00:15:23 --> 00:15:27 devices that I'll show you in a minute. 195 00:15:27 --> 00:15:30 I'm going to show you a series of three movies that were made by a 196 00:15:30 --> 00:15:33 very talent MIT graduate student, Kevin Eggan who is now a fellow over 197 00:15:33 --> 00:15:37 at Harvard, in Professor Rudolf Jaenisch's lab, 198 00:15:37 --> 00:15:40 one of my colleagues over at the Whitehead Institute. 199 00:15:40 --> 00:15:44 And the first movie is going to be the isolation of nuclei 200 00:15:44 --> 00:15:51 from somatic cells. 201 00:15:51 --> 00:15:55 So here is a glass pipette, and Kevin is drawing up a cell into 202 00:15:55 --> 00:15:59 the pipette. And he is going to spit out, he has, you'll 203 00:15:59 --> 00:16:03 see it again. He has spit out the contents of the 204 00:16:03 --> 00:16:07 cell other than the nucleus. So there's this little thing in 205 00:16:07 --> 00:16:11 there that's a nucleus. So here's another cell. 206 00:16:11 --> 00:16:15 He's pipetting it up and down to break it open and is retaining the 207 00:16:15 --> 00:16:19 nucleus because it fits well in the pipette and doesn't come out as 208 00:16:19 --> 00:16:23 readily as the other cell contents. There goes the cell into the 209 00:16:23 --> 00:16:27 pipette, and here comes the cell debris, and in the pipette is 210 00:16:27 --> 00:16:33 retained the nucleus. OK. Step two, removing the chromosomes 211 00:16:33 --> 00:16:39 from an unfertilized egg. This thing is a suction device that 212 00:16:39 --> 00:16:45 sucks and holds the mouse egg. Here is a microinjection needle. 213 00:16:45 --> 00:16:51 And it's not pointed because it's actually being helped by an electric 214 00:16:51 --> 00:16:57 pulse to enter the egg. And Kevin is now pulling out the 215 00:16:57 --> 00:17:02 metaphase plate from an egg. Those are the chromosomes from the 216 00:17:02 --> 00:17:07 egg. How does he know where they are? Well, he's a smart guy. 217 00:17:07 --> 00:17:11 And if you've looked at enough mouse eggs, you can actually see 218 00:17:11 --> 00:17:16 them under the right microscopy conditions. OK. 219 00:17:16 --> 00:17:21 So there goes the pipette again and here comes some more chromosomes. 220 00:17:21 --> 00:17:26 And there you go. OK. Easy when you know how. 221 00:17:26 --> 00:17:37 There. OK. So once you've got your 222 00:17:37 --> 00:17:41 bucket load of eggs that are enucleated and you've got your 223 00:17:41 --> 00:17:45 pipette full of somatic cell nuclei, you can put the two together. So 224 00:17:45 --> 00:17:49 here's your enucleated egg held on by suction and here's your pipette 225 00:17:49 --> 00:17:53 that has got those nuclei that came from the somatic cells. 226 00:17:53 --> 00:17:57 Watch the barrel of the pipette or the microinjection pipette 227 00:17:57 --> 00:18:02 and you will see it. There is goes. 228 00:18:02 --> 00:18:06 There's a nucleus going down into the cell. And if you watch 229 00:18:06 --> 00:18:10 carefully we will see when the pipette comes out. 230 00:18:10 --> 00:18:15 You really have got to get it out of there. That nucleus is gone. 231 00:18:15 --> 00:18:19 OK. Here's another one. There's the nucleus. You've got to watch. 232 00:18:19 --> 00:18:23 There it goes. You can actually see it going it out if you look 233 00:18:23 --> 00:18:28 carefully. He's really making sure he pushes it out. 234 00:18:28 --> 00:18:33 And there, another nucleus somatically transferred. 235 00:18:33 --> 00:18:38 OK. And the third one here is the nucleus. A little pulse of electric 236 00:18:38 --> 00:18:43 current to get the pipette in, and then you push the nucleus out by 237 00:18:43 --> 00:18:49 pressure, and there you are. And the last one, here's a lost 238 00:18:49 --> 00:19:03 nucleus. 239 00:19:03 --> 00:19:09 There is goes. All right. OK. 240 00:19:09 --> 00:19:18 So this works. It does work. 241 00:19:18 --> 00:19:22 These mice are living proof that somatic cell nuclear transfer gives 242 00:19:22 --> 00:19:26 clones and so is Dolly, but there are issues. There are 243 00:19:26 --> 00:19:35 many, many problems. 244 00:19:35 --> 00:19:39 Cloning by somatic cell nuclear transfer is very inefficient. 245 00:19:39 --> 00:19:48 It depends somewhat on the species 246 00:19:48 --> 00:19:52 one has looked at, but it is generally around or less 247 00:19:52 --> 00:19:57 than 1%. So fewer than one in a hundred of those mouse eggs actually 248 00:19:57 --> 00:20:02 make it on to give you a mouse. The ones that do, 249 00:20:02 --> 00:20:08 although they might look normal, turn out all to have something wrong 250 00:20:08 --> 00:20:13 with them. So in mice 100% of the animals that you get out have got 251 00:20:13 --> 00:20:19 something wrong with them. And this is probably true in all 252 00:20:19 --> 00:20:24 clones that have been made. It was true of Dolly who died an 253 00:20:24 --> 00:20:30 early death due to arthritis and various other issues. 254 00:20:30 --> 00:20:34 And it's true in essentially every animal, in all animals that have 255 00:20:34 --> 00:20:38 been carefully examined. So what are the problems? 256 00:20:38 --> 00:20:42 This is to remind you again that cloning efficiency from adult nuclei 257 00:20:42 --> 00:20:47 is very low. One of the problems, and these animals would not be 258 00:20:47 --> 00:20:51 viable, is something called large offspring syndrome. 259 00:20:51 --> 00:20:55 This is a normal newborn mouse and this is a cloned mouse. 260 00:20:55 --> 00:20:59 And you can see that it's at least double the size of 261 00:20:59 --> 00:21:04 the normal mouse. This is the placenta from the normal 262 00:21:04 --> 00:21:08 mouse and from the cloned mouse also enormous. So there is an issue here 263 00:21:08 --> 00:21:13 with the size of embryos or the size of the animals. 264 00:21:13 --> 00:21:17 There's also an issue with lifespan. This is a survival curve for mice. 265 00:21:17 --> 00:21:22 And mice cloned animals are pretty much all dead by about 750 days 266 00:21:22 --> 00:21:27 after birth. Whereas, for most wild type or even animals 267 00:21:27 --> 00:21:31 made by artificial reproductive technologies, your survival is way, 268 00:21:31 --> 00:21:36 way longer than at. At least double that time. 269 00:21:36 --> 00:21:41 So there is an issue with survival in general. And I'll show you a 270 00:21:41 --> 00:21:47 little bit, I pulled out a couple of panels of some primary research data 271 00:21:47 --> 00:21:52 to show you that the reason these animals are abnormal is because 272 00:21:52 --> 00:21:57 their gene expression is abnormal. OK? So this is a PCR-based assay 273 00:21:57 --> 00:22:03 looking at various RNAs from various different genes. 274 00:22:03 --> 00:22:07 OK? These are, each of these names up here is a 275 00:22:07 --> 00:22:11 gene name. It doesn't matter what they are. And each of these white 276 00:22:11 --> 00:22:15 bands here is the demonstration that an RNA is present. 277 00:22:15 --> 00:22:19 In a normal embryo there is a band under each of these gene names 278 00:22:19 --> 00:22:23 indicating that the RNA is present. In a cloned embryo, and this is 279 00:22:23 --> 00:22:27 true for all cloned embryos, you see that at least three bands 280 00:22:27 --> 00:22:31 are missing. And here's one I didn't diagram, 281 00:22:31 --> 00:22:35 didn't put an arrow on. Four bands are missing. And in different 282 00:22:35 --> 00:22:40 cloned embryos different RNAs are missing. So you can show that in 283 00:22:40 --> 00:22:45 cloned embryos where you look gene expression is abnormal. 284 00:22:45 --> 00:22:49 Why is gene expression abnormal? Well, there are a couple of reasons. 285 00:22:49 --> 00:22:54 One is that in fact we're wrong and that the DNA content of cells really 286 00:22:54 --> 00:23:00 is changing as they get older. But that's not supported by projects 287 00:23:00 --> 00:23:06 like the Human Genome Project which suggests that the genes, 288 00:23:06 --> 00:23:12 in fact, are stable in all cells. A much more plausible and accepted 289 00:23:12 --> 00:23:19 explanation of the data has to do with the control of gene expression. 290 00:23:19 --> 00:23:25 And an aspect of the control of gene expression that we have 291 00:23:25 --> 00:23:31 mentioned, although not by this particular term. 292 00:23:31 --> 00:23:38 And so I want to introduce you to a new term which is epigenetics. 293 00:23:38 --> 00:23:44 Epigenetics means outside genetics. 294 00:23:44 --> 00:23:46 And this refers to the control of gene expression that does not 295 00:23:46 --> 00:23:49 directly bear on the DNA sequence. We can have an argument about this 296 00:23:49 --> 00:23:51 later, but that's how I'm going to define it now. 297 00:23:51 --> 00:23:54 This refers to the regulation of gene expression -- 298 00:23:54 --> 00:24:03 -- that is not base 299 00:24:03 --> 00:24:17 sequence directed. 300 00:24:17 --> 00:24:23 That is not directed by the DNA base sequence. All right. 301 00:24:23 --> 00:24:29 And I wrote it for you. Let's look at some examples. 302 00:24:29 --> 00:24:33 This is a cloned cat. This is a kitten and this is its 303 00:24:33 --> 00:24:37 mom. And you can see they're very cute but they are different from one 304 00:24:37 --> 00:24:42 another. OK? They're different but they've got exactly the same DNA 305 00:24:42 --> 00:24:46 content. They're different. They're expressing different genes. 306 00:24:46 --> 00:24:51 OK? This is where we've been. Let's move somewhere else. 307 00:24:51 --> 00:24:55 I would think we might have someone who is one of an identical twin pair 308 00:24:55 --> 00:25:00 in the class. Anyone an identical twin? 309 00:25:00 --> 00:25:03 Oh, yeah. Well, we're statistically borderline. 310 00:25:03 --> 00:25:07 They're one in a thousand. So we don't have it. 311 00:25:07 --> 00:25:11 Do we have fraternal twins? Are you a fraternal twin? One. 312 00:25:11 --> 00:25:15 OK. All right. A few. OK. So there we're OK. 313 00:25:15 --> 00:25:19 All right. OK. Identical twins. 314 00:25:19 --> 00:25:23 Identical twins are generally not absolutely identical. 315 00:25:23 --> 00:25:27 They might look a bit different, they might have different behaviors, 316 00:25:27 --> 00:25:31 and they may have different more quantitative traits. 317 00:25:31 --> 00:25:35 The term concordance refers to whether or not twins share a trait. 318 00:25:35 --> 00:25:39 And that's particularly useful in looking to see whether diseases are 319 00:25:39 --> 00:25:44 causes by DNA based mutations. There's something called 320 00:25:44 --> 00:25:48 scleroderma which is a skin disorder that has 98% concordance. 321 00:25:48 --> 00:25:53 If one twin has it the other twin has it. And it is directed by 322 00:25:53 --> 00:25:57 specific changes in the DNA sequence. On the other hand, 323 00:25:57 --> 00:26:02 asthma only has a 54% concordance. And that suggests that asthma is 324 00:26:02 --> 00:26:06 directed, this is a high concordance, it's higher than fraternal twins 325 00:26:06 --> 00:26:10 have, but it certainly doesn't reach the heights of scleroderma. 326 00:26:10 --> 00:26:14 And what the data suggests is that scleroderma is caused only due to 327 00:26:14 --> 00:26:19 changes in DNA base sequence and asthma is caused due to changes in 328 00:26:19 --> 00:26:23 the DNA base sequence as well as something else, 329 00:26:23 --> 00:26:27 and the something else is believed to be changes in gene expression 330 00:26:27 --> 00:26:31 that are regulated above the level, at a different level than the base 331 00:26:31 --> 00:26:35 sequence per se. Now, if we think back to molecular 332 00:26:35 --> 00:26:39 biology and the regulation of transcription, 333 00:26:39 --> 00:26:42 I pulled this slide from one of your previous handouts, 334 00:26:42 --> 00:26:46 your gene, your promoter and your promoter activity leading to 335 00:26:46 --> 00:26:50 production of RNA. That is the level of gene 336 00:26:50 --> 00:26:53 expression that seems to go wrong in cloned embryos. 337 00:26:53 --> 00:26:57 We talked about regulation of transcription by binding of 338 00:26:57 --> 00:27:01 transcription factors to the promoter. 339 00:27:01 --> 00:27:06 And you've just had an exam on this previously. And also by something 340 00:27:06 --> 00:27:11 called chromatin modification. And epigenetics has to do with 341 00:27:11 --> 00:27:16 chromatin. Again, this is an old slide. 342 00:27:16 --> 00:27:21 You've had it previously. And I pointed out to you that DNA 343 00:27:21 --> 00:27:26 is packaged into tightly wound coils called chromatin by winding around 344 00:27:26 --> 00:27:31 particular proteins. And the particular proteins it winds 345 00:27:31 --> 00:27:35 around specifically are called histones, and these histones and the 346 00:27:35 --> 00:27:39 DNA wound around them are inhibitory to transcription and they must be 347 00:27:39 --> 00:27:43 loosened up or removed to allow transcription to take place. 348 00:27:43 --> 00:27:47 So bear that in mind while I tell you something else that can happen 349 00:27:47 --> 00:27:51 to DNA. DNA can be covalently modified, and this is on your 350 00:27:51 --> 00:27:55 handout. This is number six on your handout. DNA can be covalently 351 00:27:55 --> 00:27:59 modified. In particular, 352 00:27:59 --> 00:28:03 cytosine, the base cytosine can have a methyl group added to its 5 353 00:28:03 --> 00:28:07 position, and it is now called 5-methylcytosine. 354 00:28:07 --> 00:28:11 And this does not change the base sequence of the DNA, 355 00:28:11 --> 00:28:14 but it does have a profound affect on whether or not the DNA is 356 00:28:14 --> 00:28:18 transcriptionally active. So let me go through a cartoon with 357 00:28:18 --> 00:28:22 you to indicate how this is, and then I'll come back to the 358 00:28:22 --> 00:28:26 relevance of this for cloned animals and their abnormalities. 359 00:28:26 --> 00:28:30 So this is number seven on your handout. 360 00:28:30 --> 00:28:34 The DNA that I've shown in black, and I've shown double-stranded DNA 361 00:28:34 --> 00:28:38 as a single line here so don't get confused, is wound around purple 362 00:28:38 --> 00:28:42 barrels of histone proteins. And this chromatin configuration 363 00:28:42 --> 00:28:46 where the DNA is wound around histone proteins has to be removed 364 00:28:46 --> 00:28:50 or has to be modified to activate transcription. 365 00:28:50 --> 00:28:54 When the histones are removed the transcription factors can do their 366 00:28:54 --> 00:28:58 thing and you can get transcription taking place. 367 00:28:58 --> 00:29:04 And it turns out that when the DNA is methylated that does not happen. 368 00:29:04 --> 00:29:10 Methylation prevents the removal of the histones from the DNA template, 369 00:29:10 --> 00:29:16 from the gene, and thereby prevents transcription from taking place from 370 00:29:16 --> 00:29:22 a gene. OK? So on the handout that I've posted on the Web, 371 00:29:22 --> 00:29:28 I have put an explanation of why methylation prevents 372 00:29:28 --> 00:29:34 histone removal. I'm not going to go through it in 373 00:29:34 --> 00:29:38 class, but you can go and look at it for your interest on the Web posted. 374 00:29:38 --> 00:29:43 OK? But what I want you to have in your minds now is that methylation 375 00:29:43 --> 00:29:48 prevents DNA removal. Now, methylation therefore, 376 00:29:48 --> 00:29:52 because it can repress transcription, is used by this body to use by the 377 00:29:52 --> 00:29:57 cell as a mechanism to regulate which genes are active 378 00:29:57 --> 00:30:02 and which cell type. So in cell type one where there's a 379 00:30:02 --> 00:30:06 gene that I have depicted as so, double-stranded and there's a 380 00:30:06 --> 00:30:11 promoter, no methylation, it's expressed. That same gene in 381 00:30:11 --> 00:30:16 cell type two might be methylated and therefore not expressed. 382 00:30:16 --> 00:30:20 This is number nine on your handout. Reciprocally gene two might be 383 00:30:20 --> 00:30:25 methylated in cell type one where it's not expressed, 384 00:30:25 --> 00:30:30 not methylated in cell type two where it is expressed and so on. 385 00:30:30 --> 00:30:34 And what you can get from this is that in a particular cell type a set 386 00:30:34 --> 00:30:39 of genes will have a characteristic methylation pattern. 387 00:30:39 --> 00:30:43 And you can get a kind of methylation profile of the entire 388 00:30:43 --> 00:30:48 genome that dictates which genes are going to be active and which are not 389 00:30:48 --> 00:30:53 active, or is one level of regulation that dictates which genes 390 00:30:53 --> 00:30:58 are active and expressed and which are not. 391 00:30:58 --> 00:31:02 There is another level of methylation that has to do with 392 00:31:02 --> 00:31:06 what's in the egg and the sperm and what methylation patterns are there. 393 00:31:06 --> 00:31:10 And it turns out that not only are there cell type specific methylation 394 00:31:10 --> 00:31:14 differences but there are gender specific differences. 395 00:31:14 --> 00:31:18 And the methylation patterns of female genes, of genes in the female 396 00:31:18 --> 00:31:22 are different than some of the methylation patterns of genes in the 397 00:31:22 --> 00:31:26 male. This particular kind of methylation difference is called 398 00:31:26 --> 00:31:31 imprinting. Here's an example. Here's a gene. 399 00:31:31 --> 00:31:35 Gene one in the egg has a particular methylation pattern. 400 00:31:35 --> 00:31:40 That same gene two, gene one in the sperm has a different methylation 401 00:31:40 --> 00:31:44 pattern. OK? And this difference makes it a so-called imprinted gene. 402 00:31:44 --> 00:31:49 Here's gene two. Also differences between the male and the female 403 00:31:49 --> 00:31:54 copies of the genes. And here's gene three which has the 404 00:31:54 --> 00:31:58 same methylation pattern in both egg and sperm and is therefore 405 00:31:58 --> 00:32:03 not imprinted. So there are these two kinds of 406 00:32:03 --> 00:32:07 methylation that take place, the cell type specific and the 407 00:32:07 --> 00:32:12 gender specific. And these all get put together into 408 00:32:12 --> 00:32:16 a complicated arrangement of changing methylation patterns during 409 00:32:16 --> 00:32:21 development. And I'm going to go through this. This is number ten on 410 00:32:21 --> 00:32:26 your handout. And I'm going to go through this as an animated cartoon. 411 00:32:26 --> 00:32:30 So here is, represented by the colors, the methylation pattern in 412 00:32:30 --> 00:32:35 the egg and in the sperm. They are imprinted with their own 413 00:32:35 --> 00:32:39 methylation patterns. During fertilization they join to 414 00:32:39 --> 00:32:43 form a zygote which goes on to make an embryo. Now, 415 00:32:43 --> 00:32:47 early on during embryogenesis some cells are set aside. 416 00:32:47 --> 00:32:51 And we looked at this with determinants in Caenorhabditis. 417 00:32:51 --> 00:32:55 Some cells are set aside to make the future germ cells. 418 00:32:55 --> 00:32:59 And very interestingly as that happens there is a demethylation of 419 00:32:59 --> 00:33:04 all the imprinting of methyl groups. 420 00:33:04 --> 00:33:08 And as those germ cells then go onto become egg or sperm new methylation 421 00:33:08 --> 00:33:12 takes place and you get the imprinted patterns put back on egg 422 00:33:12 --> 00:33:17 and sperm appropriately. It's a complicated process. 423 00:33:17 --> 00:33:21 And if you think about it a little you'll see there are some real 424 00:33:21 --> 00:33:26 issues there, but I'm throwing this out as a cartoon. 425 00:33:26 --> 00:33:30 Also in the embryo new methylation patterns arise that I've called 426 00:33:30 --> 00:33:34 embryonic methylation patterns. So there are enzymes that are adding 427 00:33:34 --> 00:33:38 these methyl groups onto cytosine, and there are a bunch of different 428 00:33:38 --> 00:33:42 enzymes. And in the embryo there are new methylation patterns that 429 00:33:42 --> 00:33:45 arise. And as the embryo goes through its thing and makes its 430 00:33:45 --> 00:33:49 different cell types, additional new methylation patterns 431 00:33:49 --> 00:33:53 arise. I've called these somatic methylation patterns. 432 00:33:53 --> 00:33:57 And they are different in the different cell types, as 433 00:33:57 --> 00:34:01 I've just shown you. So there is this complicated way of 434 00:34:01 --> 00:34:06 changing methylation patterns during development, and this has 435 00:34:06 --> 00:34:12 implications for the chromatin structure and it regulates the 436 00:34:12 --> 00:34:17 expression of genes in different cell types. It is one important 437 00:34:17 --> 00:34:22 level of gene regulation. And for cloning purposes you should 438 00:34:22 --> 00:34:28 know that adult methylation patterns never normally revert to 439 00:34:28 --> 00:34:33 embryonic patterns. The germ cells are set aside very 440 00:34:33 --> 00:34:38 early during embryogenesis, and once that has happened you go on 441 00:34:38 --> 00:34:43 and make all these new methylation patterns and you never turn them 442 00:34:43 --> 00:34:48 back. OK. So what does this have to do with cloning? 443 00:34:48 --> 00:34:54 Well, the problem with clones and the problem with gene expression in 444 00:34:54 --> 00:34:59 clones, it is believed, is that there is a problem with 445 00:34:59 --> 00:35:04 changing the methylation patterns and the chromatin structure back to 446 00:35:04 --> 00:35:09 the embryonic structure. So in normal developments, 447 00:35:09 --> 00:35:13 I've summarized the cartoon that I just showed you in complicated form, 448 00:35:13 --> 00:35:18 you move from some kind of egg-sperm methylation state that moves on, 449 00:35:18 --> 00:35:23 that allows you to move on to activate the early embryonic genes. 450 00:35:23 --> 00:35:27 And because you have put in the appropriate embryonic methylation 451 00:35:27 --> 00:35:32 patterns. And then in a stepwise way you go on to act two, 452 00:35:32 --> 00:35:37 form the adult methylation patterns and activate the adult genes. 453 00:35:37 --> 00:35:42 OK? So there's a connection between the methylation patterns and correct 454 00:35:42 --> 00:35:47 gene expressions. This is a summary of the big 455 00:35:47 --> 00:35:52 cartoon I just showed you. After somatic cell nuclear transfer 456 00:35:52 --> 00:35:57 there are three possible outcomes with regard to methylation patterns 457 00:35:57 --> 00:36:02 and with the prognosis for the health of the resulting embryo. 458 00:36:02 --> 00:36:07 One outcome is that this adult donor nucleus that has its adult 459 00:36:07 --> 00:36:12 methylation patterns, when injected into enucleated egg, 460 00:36:12 --> 00:36:17 will undergo some kind of reprogramming and will land up with 461 00:36:17 --> 00:36:22 a normal embryonic set of methylation patterns. 462 00:36:22 --> 00:36:27 If it does that it will be able to support normal development. 463 00:36:27 --> 00:36:32 However, it is believed that this very, very rarely, probably 464 00:36:32 --> 00:36:36 never happens. What is much more likely is that 465 00:36:36 --> 00:36:40 this adult donor nucleus, when injected into an enucleated egg, 466 00:36:40 --> 00:36:43 goes on and is partially reprogrammed by the enzymes that are 467 00:36:43 --> 00:36:47 present in the egg. And it goes on to set up some kind 468 00:36:47 --> 00:36:51 of abnormal set of methylation patterns, some of which look like 469 00:36:51 --> 00:36:54 the embryonic patterns, some of which look like the adult 470 00:36:54 --> 00:36:58 patterns. And it goes on correspondeningly to activate gene 471 00:36:58 --> 00:37:02 expression abnormally. And, therefore, 472 00:37:02 --> 00:37:06 to make an abnormal embryo. And then a third outcome is that 473 00:37:06 --> 00:37:11 you just don't reprogram this nucleus at all, 474 00:37:11 --> 00:37:16 and it's an adult nucleus sitting in the egg. And in that case you get 475 00:37:16 --> 00:37:20 no embryo resulting from the somatic cell nuclear transfer at all. 476 00:37:20 --> 00:37:25 So it is, and this is number 12 on your slide, on your handout. 477 00:37:25 --> 00:37:29 So the notion now is that abnormal clones are abnormal because of an 478 00:37:29 --> 00:37:34 inability to reprogram the DNA, they methylation patterns and the 479 00:37:34 --> 00:37:39 chromatin structure to turn you into a normal embryo. 480 00:37:39 --> 00:37:44 So let's move on to the final thing I want to discuss with you today 481 00:37:44 --> 00:37:49 which are issues. Issues with somatic cell nuclear 482 00:37:49 --> 00:37:55 transfer and the whole notion of cloning. And I have a list of them 483 00:37:55 --> 00:38:00 here. They fall into two categories. One are the ethical issues and the 484 00:38:00 --> 00:38:06 other are the methodological issues. 485 00:38:06 --> 00:38:09 We raised last time the ethics of using human embryos to make human 486 00:38:09 --> 00:38:13 stem cell lines. And this ethical issue is still 487 00:38:13 --> 00:38:17 there. Is it acceptable to take a human embryo at the blastocystic 488 00:38:17 --> 00:38:21 stage, when it's a ball of cells and to harvest it, 489 00:38:21 --> 00:38:25 to kill it, to take the cells and to make stem cell lines? 490 00:38:25 --> 00:38:29 Someone asked me whether you couldn't take one cell out of that 491 00:38:29 --> 00:38:33 embryo and make a cell line out of that embryo and let the rest 492 00:38:33 --> 00:38:37 of the embryo grow. And in theory you actually could do 493 00:38:37 --> 00:38:41 that. It would be a pain in the neck, but you actually in theory 494 00:38:41 --> 00:38:45 could do that. OK? But that's not a possibility 495 00:38:45 --> 00:38:49 that's really being seriously entertained. This whole use of 496 00:38:49 --> 00:38:53 human embryos to support actually making a whole organism has its real 497 00:38:53 --> 00:38:57 issues. What if you do that and you land up with as you do in the mice 498 00:38:57 --> 00:39:02 and the cows and the sheep and the everything else? 499 00:39:02 --> 00:39:06 You land up with a whole bunch of abnormal human beings, 500 00:39:06 --> 00:39:10 what are you going to do about this? What's the fate of them, what's 501 00:39:10 --> 00:39:14 your commitment to these abnormal babies and what is the commitment of 502 00:39:14 --> 00:39:18 society here? These are very big issues. Another issue, 503 00:39:18 --> 00:39:22 which is a very real one, is where are the eggs that you're 504 00:39:22 --> 00:39:26 going to do all this somatic cell nuclear transfer going to come from? 505 00:39:26 --> 00:39:30 So there was a recent report from a Korean group who had actually made a 506 00:39:30 --> 00:39:34 human stem cell line after somatic cell nuclear transfer. 507 00:39:34 --> 00:39:38 They had used 650 human eggs. And they had used 16 women to 508 00:39:38 --> 00:39:43 donate these 650 eggs. At the present rate of making 509 00:39:43 --> 00:39:48 clones and making stem cell lines, you would probably need at least 500 510 00:39:48 --> 00:39:53 to 1,000 human eggs per attempt to get autologous stem cells. 511 00:39:53 --> 00:39:57 Now, I told you previously, when we talked about assisted 512 00:39:57 --> 00:40:02 reproductive technology and you retrieve oocytes from the ovary and 513 00:40:02 --> 00:40:07 do in vitro fertilization, you usually get about 20 oocytes 514 00:40:07 --> 00:40:12 every time you try to get eggs from a donor. 515 00:40:12 --> 00:40:16 That's a lot of people who have to donate these eggs. 516 00:40:16 --> 00:40:20 And if you're actually going to try and grow these into babies that's a 517 00:40:20 --> 00:40:24 lot of surrogate mothers. So this is an issue that has not 518 00:40:24 --> 00:40:28 been resolved or even discussed really very well. 519 00:40:28 --> 00:40:32 But these are the ethical issues that make these scientific issues 520 00:40:32 --> 00:40:36 move into the realm of legislature. There is currently a federal ban on 521 00:40:36 --> 00:40:40 using federal money to make human stem cell lines. 522 00:40:40 --> 00:40:44 So you cannot use money from the National Institutes of Health to do 523 00:40:44 --> 00:40:48 this. There is no actual ban on trying to clone humans per se. 524 00:40:48 --> 00:40:52 There is a gridlock because it's not clear whether to separate 525 00:40:52 --> 00:40:56 therapeutic and reproductive cloning from one another, 526 00:40:56 --> 00:41:01 but these are issues that are very loaded. 527 00:41:01 --> 00:41:05 And, as I mentioned last time, in the Massachusetts House we just 528 00:41:05 --> 00:41:09 approved the use of private funding to make human stem cell lines in 529 00:41:09 --> 00:41:13 Massachusetts, but not the use of federal funding. 530 00:41:13 --> 00:41:18 We cannot do that. OK. Methods. To date no primate has been cloned. 531 00:41:18 --> 00:41:22 There is something difficult about cloning primates. 532 00:41:22 --> 00:41:26 No human, no monkey has been cloned yet. Why? What's the problem? 533 00:41:26 --> 00:41:30 Not clear at this point, but I suspect those problems will be 534 00:41:30 --> 00:41:35 overcome in the near future. OK. Some other thoughts. 535 00:41:35 --> 00:41:39 What about these eggs? I told you the source of eggs for somatic cell 536 00:41:39 --> 00:41:43 nuclear transfer in humans is a big issue. What if you could turn ES 537 00:41:43 --> 00:41:47 cells into eggs? Could you treat then with something 538 00:41:47 --> 00:41:51 and turn them into eggs? And that would overcome the need 539 00:41:51 --> 00:41:55 for donors. Well, in fact, an experiment was published 540 00:41:55 --> 00:41:59 a little while ago showing that you could take ES cells and you could 541 00:41:59 --> 00:42:04 activate them in various ways. And activate expression of these 542 00:42:04 --> 00:42:09 proteins that you should recognize, ZP1, ZP2 and ZP3. Remember those? 543 00:42:09 --> 00:42:13 Yes? OK, the zona pellucida proteins. So that you could take 544 00:42:13 --> 00:42:18 these cells and at least start turning them into eggs. 545 00:42:18 --> 00:42:23 That's the sense that you might be able to do. OK. 546 00:42:23 --> 00:42:28 And then finally the question of how can you reprogram these adult 547 00:42:28 --> 00:42:33 nuclei to make the whole process more efficient? 548 00:42:33 --> 00:42:37 And I'll end with telling you two thoughts that are being entertained 549 00:42:37 --> 00:42:42 by people in the field. One of them is to take an adult 550 00:42:42 --> 00:42:46 donor nucleus and to try to reprogram it into an embryonic state 551 00:42:46 --> 00:42:51 sequentially. So if you take the adult donor nucleus -- 552 00:42:51 --> 00:42:59 Thank you. If you take the adult 553 00:42:59 --> 00:43:03 donor nucleus, you inject it into an enucleated egg, 554 00:43:03 --> 00:43:07 you let it partially reprogram, and then you take the nucleus out of 555 00:43:07 --> 00:43:11 that partial embryo and put it into a new egg, that nucleus does much, 556 00:43:11 --> 00:43:15 much better. And that's true in frogs as well. 557 00:43:15 --> 00:43:19 And you can then sequentially reactivate the genetic program. 558 00:43:19 --> 00:43:23 And that's actually one of the best arguments that the genetic material 559 00:43:23 --> 00:43:27 really does stay the same. And it's a problem with the 560 00:43:27 --> 00:43:31 chromatin structure of the DNA. And the other dream that people are 561 00:43:31 --> 00:43:35 pursuing is to take these adult donor nuclei and incubate them in 562 00:43:35 --> 00:43:40 some kind of reprogramming extract, some mix of proteins that will 563 00:43:40 --> 00:43:44 magically change the chromatin structure of the nucleus and allow 564 00:43:44 --> 00:43:49 it to revert to a normal embryonic nucleus so it an go on and do its 565 00:43:49 --> 00:43:52 thing. And I am going to stop there. And thank you very much.