1 00:02:57 --> 00:03:01 OK. So last time I reviewed for you some of the basic cloning techniques. 2 00:03:01 --> 00:03:05 I failed to show you this slide, which I intended to do, which is a 3 00:03:05 --> 00:03:09 commercially available plasmid. I told you about plasmids last time, 4 00:03:09 --> 00:03:14 small circular DNA molecules that are used in the purpose of cloning. 5 00:03:14 --> 00:03:18 They're derived from nature, but then they've been heavily 6 00:03:18 --> 00:03:23 manipulated by scientists to be useful for the purposes of cloning. 7 00:03:23 --> 00:03:27 Recall that they have two, three critical elements, an origin 8 00:03:27 --> 00:03:31 of replication to allow them to be replicated inside of bacteria, 9 00:03:31 --> 00:03:35 a selectable marker, a drug-resistance gene, 10 00:03:35 --> 00:03:39 ampicillin resistance gene for example, and finally, 11 00:03:39 --> 00:03:43 can you now show the middle slide? He's working on it. And finally 12 00:03:43 --> 00:03:47 what we call a multiple cloning site, a set of restriction sites, 13 00:03:47 --> 00:03:51 restriction enzyme recognition sites that allow us to pop 14 00:03:51 --> 00:03:56 in pieces of DNA. I have dual paper readers. 15 00:03:56 --> 00:04:01 Dual paper readers? That doesn't happen very often. 16 00:04:01 --> 00:04:05 You usually have one but not often two in a row. Good. 17 00:04:05 --> 00:04:10 So these are useful practical tools. Maybe before I forget, 18 00:04:10 --> 00:04:15 I'll mention, remind you of the quiz on Monday, where you go based on 19 00:04:15 --> 00:04:20 your last name. There was a discussion last time 20 00:04:20 --> 00:04:25 about the review session, which is actually not on here but we 21 00:04:25 --> 00:04:30 didn't change the review session. Oh, because it was last night. 22 00:04:30 --> 00:04:34 We weren't able to change the review session, which was last night, 23 00:04:34 --> 00:04:38 but there is a tutoring session which will cover the same material 24 00:04:38 --> 00:04:42 from today between 4:00 and 6:00. And then there are additional 25 00:04:42 --> 00:04:46 sessions with the TAs, including up till Monday morning 26 00:04:46 --> 00:04:50 before the exam. So there should be ample 27 00:04:50 --> 00:04:54 opportunity to get your questions answered. So plasmids are now sort 28 00:04:54 --> 00:04:58 of a useful and are a very available tool. 29 00:04:58 --> 00:05:02 This figure comes from your book. It's a version of what we covered 30 00:05:02 --> 00:05:06 in our example last time, the cloning of the T gene. 31 00:05:06 --> 00:05:10 Here in red is the starting material, the DNA from the source. 32 00:05:10 --> 00:05:15 In our case it was S. pyogenes, that flesh eating bacterium, 33 00:05:15 --> 00:05:19 but it could be anything. It could be genomic DNA from you. 34 00:05:19 --> 00:05:23 You take that genomic DNA, whatever the source material is, 35 00:05:23 --> 00:05:27 you digest it, cut it with a restriction enzyme, 36 00:05:27 --> 00:05:32 with a particular restriction enzyme. You then mix that DNA with plasmid 37 00:05:32 --> 00:05:37 DNA which has been cut with the same restriction enzyme, 38 00:05:37 --> 00:05:42 the sticky ends of the linear molecule anneal with the sticky ends 39 00:05:42 --> 00:05:47 of the plasmid. You then add DNA ligase to seal 40 00:05:47 --> 00:05:52 those nicks to produce full covalently closed circular molecules 41 00:05:52 --> 00:05:57 that are composed of both the plasmid and now an insert 42 00:05:57 --> 00:06:02 from the DNA sample. You then take those recombinant 43 00:06:02 --> 00:06:06 plasmids, mix them with bacteria under conditions which allow the DNA 44 00:06:06 --> 00:06:10 to get into those bacterial cells. This is a process called 45 00:06:10 --> 00:06:14 transformation. You then plate those bacteria onto 46 00:06:14 --> 00:06:19 auger plates that contain the antibiotic, in this case ampicillin 47 00:06:19 --> 00:06:23 in our example. The bacteria that didn't get a 48 00:06:23 --> 00:06:27 plasmid die. The bacteria that did get a plasmid survive and form 49 00:06:27 --> 00:06:32 colonies on the dish. And then the question I left you 50 00:06:32 --> 00:06:36 with was how are we going to find the colony or colonies that carry 51 00:06:36 --> 00:06:41 the T gene? How are we going to find the gene, 52 00:06:41 --> 00:06:45 the recombinant plasmid, the colony carrying the recombinant 53 00:06:45 --> 00:06:49 plasmid with the T gene? I also want to mention a bit of 54 00:06:49 --> 00:06:54 terminology, which we will come back to later. We often refer to the 55 00:06:54 --> 00:06:58 collection of colonies, the collection of recombinant 56 00:06:58 --> 00:07:03 plasmids contained within those bacteria as a library. 57 00:07:03 --> 00:07:07 Like a library containing books, this is a collection of different 58 00:07:07 --> 00:07:11 things. In this case, different recombinant plasmids which 59 00:07:11 --> 00:07:15 you can go back to repeatedly taking isolates from the colony. 60 00:07:15 --> 00:07:19 In our case we would then grow up the individual bacteria to get ample 61 00:07:19 --> 00:07:23 amounts of the plasmid of interest. And this term library is used in 62 00:07:23 --> 00:07:27 molecular biology a lot, a diverse collection of clone 63 00:07:27 --> 00:07:32 fragments to which you can return depending on your specific needs. 64 00:07:32 --> 00:07:38 So how are we going to isolate our plasmid of interest? 65 00:07:38 --> 00:07:44 Well, if you recall, we knew the sequence of the genome 66 00:07:44 --> 00:07:50 of S. pyogenes. And, therefore, 67 00:07:50 --> 00:07:57 we knew the sequence of the T gene. So imagine that the T gene had a 68 00:07:57 --> 00:08:03 particular sequence starting from the 5 prime end of 69 00:08:03 --> 00:08:10 A-G-G-C-T-G-G-T-G-G-G-A to the 3 prime end. 70 00:08:10 --> 00:08:14 So this is imbedded within the T gene. The reverse complement on the 71 00:08:14 --> 00:08:19 other strand reading in this direction from the 5 prime end would 72 00:08:19 --> 00:08:23 be T, did I say that, what's this? C-C-C-A-C-C-A-G-C-C-T 73 00:08:23 --> 00:08:28 3 prime. OK? So we know a bit about the thing we're 74 00:08:28 --> 00:08:34 interested in. We know its sequence. 75 00:08:34 --> 00:08:40 And we can use this information to our advantage to isolate the plasmid 76 00:08:40 --> 00:08:47 that carries this fragment. So the bacteria, as shown on this 77 00:08:47 --> 00:08:53 slide, are present in colonies. Each colony contains a recombinant 78 00:08:53 --> 00:09:00 plasmid. The first step in isolating the plasmid of interest is 79 00:09:00 --> 00:09:06 to make a copy of the plate that carries these bacteria to replicate 80 00:09:06 --> 00:09:13 what's on this plate onto a piece of filter paper. 81 00:09:13 --> 00:09:20 We call this replica plating. 82 00:09:20 --> 00:09:27 And in this case we're going to do 83 00:09:27 --> 00:09:33 it onto a filter, a piece of paper or nylon to make a 84 00:09:33 --> 00:09:39 copy of the colonies and the pattern of the colonies onto something else 85 00:09:39 --> 00:09:45 that we can manipulate. So we make an exact copy. 86 00:09:45 --> 00:09:51 And this done by literally placing the piece of filter paper onto the 87 00:09:51 --> 00:09:57 Petri dish, as shown here. Here is the Petri dish with the 88 00:09:57 --> 00:10:01 colonies. You place a filter on top of those 89 00:10:01 --> 00:10:04 colonies. They'll stick to it. Some of the cells in those colonies 90 00:10:04 --> 00:10:08 will stick to the filter. You then pull the filter off and 91 00:10:08 --> 00:10:11 you get some of the colonies, some of the cells sticking in 92 00:10:11 --> 00:10:14 exactly the place where they were on the original plate. 93 00:10:14 --> 00:10:17 You can actually place this on another plate and allow those cells 94 00:10:17 --> 00:10:21 to grow directly on the piece of filter paper. So you make an exact 95 00:10:21 --> 00:10:24 copy of what was here onto a piece of paper. Now, 96 00:10:24 --> 00:10:28 what are you going to do with that? Well, we're going to use this 97 00:10:28 --> 00:10:33 information that we have about the sequence of the gene to isolate the 98 00:10:33 --> 00:10:38 colony of interest. First thing we do is to lyse the 99 00:10:38 --> 00:10:44 bacteria that are now present on this filter and denature the DNA. 100 00:10:44 --> 00:10:50 What I mean by denature the DNA is 101 00:10:50 --> 00:10:54 to pull it apart. It's double-stranded when it's 102 00:10:54 --> 00:10:58 present in the bacteria. To denature DNA is to make it 103 00:10:58 --> 00:11:02 single-stranded. And typically the way we do that in 104 00:11:02 --> 00:11:06 these applications is to place a piece of filter paper under high pH 105 00:11:06 --> 00:11:11 conditions, and it causes the DNA strands to melt apart. 106 00:11:11 --> 00:11:16 So now the plasmids, which were double-stranded in these bacterial 107 00:11:16 --> 00:11:20 cells, were double-stranded here have now been pulled apart into 108 00:11:20 --> 00:11:25 single-stranded molecules. So, on this piece of filter paper 109 00:11:25 --> 00:11:30 there are cells with unwound DNA, single-stranded DNA in them in this 110 00:11:30 --> 00:11:35 same pattern, which I won't try to reproduce here. 111 00:11:35 --> 00:11:40 And the question is how are we going to identify which colony 112 00:11:40 --> 00:11:45 carries a piece of the T gene? How are we going to do that? Does 113 00:11:45 --> 00:11:50 anybody have a clue? All you need to know is present up 114 00:11:50 --> 00:12:01 here. Yeah? 115 00:12:01 --> 00:12:04 So you're thinking of the example from the book in which the, 116 00:12:04 --> 00:12:08 in the example in the book they used a plasmid that had two selectable 117 00:12:08 --> 00:12:12 markers. And you were interested in those that grew under one antibiotic 118 00:12:12 --> 00:12:16 selection and didn't grow under a different antibiotic selection. 119 00:12:16 --> 00:12:20 In our case, in our plasmid, we actually didn't have two selectable 120 00:12:20 --> 00:12:24 markers. We only had one. So we're not going to use that 121 00:12:24 --> 00:12:28 strategy. We have to rely on some of the information I've 122 00:12:28 --> 00:12:32 given you already. Well, we know the sequence of the T 123 00:12:32 --> 00:12:37 gene. We know, for example, this little bit of the 124 00:12:37 --> 00:12:42 T gene, so we can make some that we call a probe. A probe, 125 00:12:42 --> 00:12:47 for this application, is an oligonucleotide, 126 00:12:47 --> 00:12:52 a small segment of nucleotides that we can synthesize. 127 00:12:52 --> 00:12:57 You can actually make any DNA sequence you want using 128 00:12:57 --> 00:13:02 chemical synthesis. This has now been fully automated. 129 00:13:02 --> 00:13:06 So you can just punch the sequence into a computer, 130 00:13:06 --> 00:13:11 attach it to one of these machines, the machine will go off and make a 131 00:13:11 --> 00:13:15 linear piece of DNA of any sequence you want. It's like a typewriter 132 00:13:15 --> 00:13:20 almost. You type it in, and what you get back out is a piece 133 00:13:20 --> 00:13:25 of DNA that is the sequence that you typed in. So we can make any DNA we 134 00:13:25 --> 00:13:29 want such that we could make a piece of DNA that had the sequence 135 00:13:29 --> 00:13:36 A-G-G-C-T-G-G-T-G-G-G-A. 136 00:13:36 --> 00:13:41 OK? We could make that piece of DNA. Moreover, we could modify that 137 00:13:41 --> 00:13:47 piece of DNA with enzymes to add a radioactive phosphate at this 5 138 00:13:47 --> 00:13:52 prime end. So we could make a radio labeled probe. 139 00:13:52 --> 00:13:58 So now that we have that, what are we going to do? Anybody? 140 00:13:58 --> 00:14:05 Yes. Yes. 141 00:14:05 --> 00:14:10 Right. 142 00:14:10 --> 00:14:14 Exactly. So this is now complementary to the other strand. 143 00:14:14 --> 00:14:18 The DNA within these cells that have been lysed and now stuck onto 144 00:14:18 --> 00:14:22 this filter are also single-stranded. So this complementary strand is 145 00:14:22 --> 00:14:26 going to be sitting in a single stranded conformation ready to 146 00:14:26 --> 00:14:30 anneal, hybridize with this probe. So you apply this radioactive probe 147 00:14:30 --> 00:14:34 to this filter. It will bind somewhat 148 00:14:34 --> 00:14:37 indiscriminately at first, but it will bind very tightly to 149 00:14:37 --> 00:14:41 that complementaryary strand from the T gene. And remember there are 150 00:14:41 --> 00:14:44 probably a million bacterial cells within this colony, 151 00:14:44 --> 00:14:48 so there will be a million copies of the probe anneal to this colony. 152 00:14:48 --> 00:14:51 You do have to do a little washing to get rid of the nonspecific 153 00:14:51 --> 00:14:55 binding which will take place. So you wash the cells, or wash the 154 00:14:55 --> 00:14:59 filter, get rid of the nonspecific binding. 155 00:14:59 --> 00:15:03 The specific binding will withstand the wash. And then you apply a 156 00:15:03 --> 00:15:07 piece of x-ray film. And what you'll end up with is a 157 00:15:07 --> 00:15:11 faint image of the filter, some very faint signal from the 158 00:15:11 --> 00:15:16 colonies that don't hybridize, and a very strong signal from the 159 00:15:16 --> 00:15:20 colonies that did hybridize properly to the probe. OK? 160 00:15:20 --> 00:15:24 So now you know, based on the position on that filter, 161 00:15:24 --> 00:15:28 which colony it is that carries the T gene. And in this case it's that 162 00:15:28 --> 00:15:33 one right there. So we can go back to that master 163 00:15:33 --> 00:15:37 plate that we've kept, use a toothpick, pluck off that 164 00:15:37 --> 00:15:41 colony and grow it up into high concentrations successfully having 165 00:15:41 --> 00:15:45 completed our cloning project of the ever toxic T gene. 166 00:15:45 --> 00:15:49 And that's gone through here in this figure, if you want to look at 167 00:15:49 --> 00:15:53 it again. You take this filter that has the colonies. 168 00:15:53 --> 00:15:57 It's actually the step here where you grow the cells up on 169 00:15:57 --> 00:16:01 the filter itself. You then lyse the cells, 170 00:16:01 --> 00:16:05 denature the DNA, add the radioactive probe. 171 00:16:05 --> 00:16:08 It won't hybridize strongly to those colonies that don't carry the 172 00:16:08 --> 00:16:12 clone of interest. It will hybridize strongly to the 173 00:16:12 --> 00:16:15 colonies that do have the clone of interest. And then through this 174 00:16:15 --> 00:16:19 x-ray film method you can visualize where those colonies are and then go 175 00:16:19 --> 00:16:23 back and pick the colonies from the master plate. OK? 176 00:16:23 --> 00:16:26 So that's how it works. It's fairly straightforward and 177 00:16:26 --> 00:16:30 works extremely well to give you the cells and clones of 178 00:16:30 --> 00:16:41 interest. Woops. 179 00:16:41 --> 00:16:46 So that's what we call clone identification by hybridization 180 00:16:46 --> 00:16:51 using a radioactive probe. And I should mention, because it 181 00:16:51 --> 00:16:57 will come up again later, that we use these radioactive probes 182 00:16:57 --> 00:17:02 extensively for molecular biology. And we use non-radioactive probes, 183 00:17:02 --> 00:17:07 oligonucleotide synthesized in the same way that I mentioned for all 184 00:17:07 --> 00:17:12 sorts of molecular biology applications in this era. 185 00:17:12 --> 00:17:17 And you'll see examples later in the lecture. I want to briefly 186 00:17:17 --> 00:17:22 mention another technique known as cloning by complementation. 187 00:17:22 --> 00:17:27 In this instance, we're going to clone by the function of the plasmid 188 00:17:27 --> 00:17:32 that we've introduced into the cells. 189 00:17:32 --> 00:17:36 Not its sequence. By its function. And the example 190 00:17:36 --> 00:17:40 I'm going to give you is to clone a gene that you might know about based 191 00:17:40 --> 00:17:44 on the biochemistry of the organism, but you haven't identified the gene 192 00:17:44 --> 00:17:49 that encodes that enzyme. The example I'm going to give you 193 00:17:49 --> 00:17:53 is to clone an enzyme which is responsible for catalyzing the 194 00:17:53 --> 00:17:57 breakdown of lactose, a disaccharide. And this is 195 00:17:57 --> 00:18:02 accomplished by an enzyme called beta-galactosidase. 196 00:18:02 --> 00:18:06 And it converts glucose. It converts lactose to glucose and 197 00:18:06 --> 00:18:11 galactose. And this is an important enzyme in bacteria and also in you. 198 00:18:11 --> 00:18:15 The enzyme is encoded by the lacZ gene of bacteria. 199 00:18:15 --> 00:18:20 OK? And this exercise that we're going to go through is to try to 200 00:18:20 --> 00:18:25 isolate the lacZ gene through this method called cloning by 201 00:18:25 --> 00:18:29 complementation. We need to use a trick, 202 00:18:29 --> 00:18:34 which I'll show you on this slide. And that is that there's an 203 00:18:34 --> 00:18:40 artificial substrate for this enzyme which is called in the field X-gal. 204 00:18:40 --> 00:18:46 It's related to lactose. The important thing is that when X-gal 205 00:18:46 --> 00:18:52 gets cleaved by beta-galactosidase, it releases galactose and this 206 00:18:52 --> 00:18:58 molecule which precipitates and turns blue. So we have a visual 207 00:18:58 --> 00:19:04 indicator of beta-galactosidase activity by placing in the medium, 208 00:19:04 --> 00:19:10 either in liquid medium or on a Petri dish, this substance X-gal. 209 00:19:10 --> 00:19:16 We know whether or not the cells have beta-galactosidase activity by 210 00:19:16 --> 00:19:23 virtue of whether the cells or the colonies turn blue. 211 00:19:23 --> 00:19:29 OK. So the first thing that we're going to do in this technique is to 212 00:19:29 --> 00:19:38 take some wild type E. coli. 213 00:19:38 --> 00:19:42 If we take a liquid culture of wild type E. coli and we plate them onto 214 00:19:42 --> 00:19:47 a tissue culture, a Petri dish that contains X-gal, 215 00:19:47 --> 00:19:52 this substrate that in the presence of beta-galactosidase will turn blue, 216 00:19:52 --> 00:19:57 will get colonies. And what color will those colonies 217 00:19:57 --> 00:20:02 be? Blue. These are normal E. 218 00:20:02 --> 00:20:07 coli. So they're able to metabolize the X-gal and produce this blue 219 00:20:07 --> 00:20:12 pigment. OK? Now, I'm interested in the enzyme, 220 00:20:12 --> 00:20:17 the gene that encodes the enzyme that carries out this activity. 221 00:20:17 --> 00:20:22 And the way that I'm going to get to it is first to isolate mutant E. 222 00:20:22 --> 00:20:27 coli. It cannot do this reaction. I'm going to try to isolate a mutant 223 00:20:27 --> 00:20:32 strain that's deficient in beta-galactosidase activity. 224 00:20:32 --> 00:20:37 And the way that I do that is to mutagenize the wild type E. 225 00:20:37 --> 00:20:42 coli. I can do this by physical methods or more commonly by just 226 00:20:42 --> 00:20:48 adding a chemical mutagen to the broth that the E. 227 00:20:48 --> 00:20:53 coli is growing in. I mutagenize the cells and then I 228 00:20:53 --> 00:20:59 plate them onto a plate that has X-gal. 229 00:20:59 --> 00:21:03 Now, most of the cells that got mutagenized will have mutated some 230 00:21:03 --> 00:21:08 other gene, not the beta-galactosidase gene, 231 00:21:08 --> 00:21:13 some other gene, or maybe no gene, such that what color will those 232 00:21:13 --> 00:21:18 colonies be? They'll be blue because they still have a functional 233 00:21:18 --> 00:21:23 beta-galactosidase enzyme. They still have a wild type lacZ 234 00:21:23 --> 00:21:28 gene. So most of the colonies will be blue. But what if the cell 235 00:21:28 --> 00:21:33 incurred a mutation in the lacZ gene that knocked out lacZ function? 236 00:21:33 --> 00:21:36 What color will the colony be? Not blue. It will be white. OK? 237 00:21:36 --> 00:21:40 And we're going to assume, for the sake of argument, 238 00:21:40 --> 00:21:44 that if you get a white colony it means that the cells have a mutation 239 00:21:44 --> 00:21:48 in this gene lacZ. That's actually not a safe 240 00:21:48 --> 00:21:52 assumption. It could have a mutation in some other gene, 241 00:21:52 --> 00:21:56 but we're not going to worry about that today. Just say that if we get 242 00:21:56 --> 00:22:00 a white colony we're going to assume that the cells have a mutation 243 00:22:00 --> 00:22:06 in the lacZ gene. So now we want to clone the lacZ 244 00:22:06 --> 00:22:15 gene. Our goal is to clone the lacZ gene. What are we going to do? 245 00:22:15 --> 00:22:25 What are we going to do? Well, I'll give you the first clue. We're 246 00:22:25 --> 00:22:33 going to make a library. We're going to make a library of 247 00:22:33 --> 00:22:39 fragments of DNA some of which will carry the lacZ gene. 248 00:22:39 --> 00:22:45 From what are we going to make that library? What will be our source 249 00:22:45 --> 00:22:51 DNA to make that library? Will it be these cells isolated 250 00:22:51 --> 00:22:57 from this colony or will it be some other cell? Do these cells have a 251 00:22:57 --> 00:23:02 functional lacZ gene? No. Do these cells? 252 00:23:02 --> 00:23:08 Yes. So you want to make a library from wild type E. 253 00:23:08 --> 00:23:13 coli. So that you have a bunch of plasmids that carry fragments of the 254 00:23:13 --> 00:23:19 E. coli genome some of which carry the lacZ gene. 255 00:23:19 --> 00:23:25 What am I going to do with that library? What am I going to do with 256 00:23:25 --> 00:23:39 that collection of clones? 257 00:23:39 --> 00:23:45 I'm going to transform it en masse all the different clones to a 258 00:23:45 --> 00:23:51 population of cells. What cells will I transform it into? 259 00:23:51 --> 00:23:57 Which bacteria? Would I put those ones into the 260 00:23:57 --> 00:24:05 lacZ mutants? 261 00:24:05 --> 00:24:10 And then I plate that transformation onto plates that contain amp. 262 00:24:10 --> 00:24:16 I need the drug to identify those cells that have picked up any 263 00:24:16 --> 00:24:21 plasmid because I don't care about cells that haven't picked up any 264 00:24:21 --> 00:24:27 plasmid so I use amp plates. And what else do the plates have in 265 00:24:27 --> 00:24:32 them? The indicator of lacZ activity, 266 00:24:32 --> 00:24:37 X-gal. So I'm going to take these transformants, 267 00:24:37 --> 00:24:42 which were derived from these cells, and I'm going to plate them out onto 268 00:24:42 --> 00:24:46 a plate. What color will most of the colonies be? 269 00:24:46 --> 00:24:51 Will most of them, raise your hand. Will most of them 270 00:24:51 --> 00:24:56 be white? Will most of them be blue? In fact, most of them will be white 271 00:24:56 --> 00:25:01 because most of the cells did not pick up a plasmid that carries the 272 00:25:01 --> 00:25:06 functional lacZ gene. Most of the cells picked up a 273 00:25:06 --> 00:25:10 plasmid that carried some other piece of the E. 274 00:25:10 --> 00:25:15 coli genome. OK? So most of the cells will not be, 275 00:25:15 --> 00:25:20 will not have functional lacZ activity, they'll be white. 276 00:25:20 --> 00:25:24 However, at some frequency a clone, a cell will pick up a recombinant 277 00:25:24 --> 00:25:29 plasmid that does carry the lacZ gene and that colony 278 00:25:29 --> 00:25:34 will turn blue. And that's the colony that we're now 279 00:25:34 --> 00:25:38 interested in because it has reconstituted lacZ activity. 280 00:25:38 --> 00:25:42 The mutant, the recombinant DNA has complemented the mutation. 281 00:25:42 --> 00:25:51 So I could isolate this plasmid and 282 00:25:51 --> 00:25:54 sequence it. And based on the sequence I might have confidence 283 00:25:54 --> 00:25:57 that indeed it is the lacZ gene. Remember I told you that there was 284 00:25:57 --> 00:26:00 an assumption built in here that this mutation really did affect the 285 00:26:00 --> 00:26:04 lacZ gene? And that's why the cells were white. 286 00:26:04 --> 00:26:07 That was an assumption. And to confirm that assumption we 287 00:26:07 --> 00:26:10 could look at the sequence of the clone that we isolated and figure it 288 00:26:10 --> 00:26:14 out, figure out whether it's the right enzyme. OK? 289 00:26:14 --> 00:26:17 So I find that teaching this is always a little confusing. 290 00:26:17 --> 00:26:20 I tried to simplify it today and go through it slowly. 291 00:26:20 --> 00:26:24 Hopefully you took notes and you can think about it on your own. 292 00:26:24 --> 00:26:27 It is relatively straightforward and it's a useful concept to 293 00:26:27 --> 00:26:30 understand that A) you could isolate mutations, and B) you can find the 294 00:26:30 --> 00:26:34 genes that were mutated by complementing those mutations 295 00:26:34 --> 00:26:44 through cloning. OK? 296 00:26:44 --> 00:26:47 OK. So now that I've spent a lecture and a half talking about 297 00:26:47 --> 00:26:51 cloning, I'm now going to tell you that cloning, while it was 298 00:26:51 --> 00:26:54 incredibly important and still is, has almost been superceded by 299 00:26:54 --> 00:26:58 another technique. It's not that we don't use cloning. 300 00:26:58 --> 00:27:02 We actually use it all the time. But it's been joined by another 301 00:27:02 --> 00:27:08 technique known as PCR which stands for the polymerase chain reaction. 302 00:27:08 --> 00:27:14 And any of you who have worked in 303 00:27:14 --> 00:27:18 molecular biology labs have been exposed to this because it is a 304 00:27:18 --> 00:27:22 totally pervasive technology. Everybody uses it. And it's not 305 00:27:22 --> 00:27:26 just in molecular biology labs. Forensic labs. Archeology labs. 306 00:27:26 --> 00:27:30 Anybody who's interested in generating large amounts of DNA from 307 00:27:30 --> 00:27:35 a small amount of DNA uses this technique called PCR. 308 00:27:35 --> 00:27:38 It was invented only a few years ago, but it's incredibly important. 309 00:27:38 --> 00:27:41 In fact, the guy who invented it, who was not a very well known 310 00:27:41 --> 00:27:45 scientist, won the Nobel Prize just a couple of years after he invented 311 00:27:45 --> 00:27:48 it because it became so powerful so quickly. It's a very, 312 00:27:48 --> 00:27:52 very simple technique. It's one of these ah-ha techniques that after 313 00:27:52 --> 00:27:55 it's been invented and described everybody says, 314 00:27:55 --> 00:27:59 oh, I could have thought of that. But, actually, nobody did until 315 00:27:59 --> 00:28:02 this guy, Kary Mullis. So I'm going to show it to you 316 00:28:02 --> 00:28:05 briefly on the board. And then we're going to go through 317 00:28:05 --> 00:28:08 a movie that shows it again. So hopefully you'll get how it 318 00:28:08 --> 00:28:12 works. Importantly, it relies on having some information 319 00:28:12 --> 00:28:15 about the DNA that you want to amplify up in large quantities. 320 00:28:15 --> 00:28:18 Again, the goal here is to amplify up a piece of DNA of interest to 321 00:28:18 --> 00:28:21 large quantities. This technique is so powerful that 322 00:28:21 --> 00:28:24 you can use a single DNA molecule to start. You can use the amount of 323 00:28:24 --> 00:28:27 DNA that you get from hair from a crime scene, for example. 324 00:28:27 --> 00:28:31 And that's mostly the way, you know. 325 00:28:31 --> 00:28:34 What is it? CSI Miami and stuff. They use this technique all the 326 00:28:34 --> 00:28:38 time. So you can use minuscule amounts of DNA. 327 00:28:38 --> 00:28:42 Even a single molecule is enough. But you do need to know a little 328 00:28:42 --> 00:28:51 bit of known sequence. 329 00:28:51 --> 00:28:54 So if you know a little bit of known sequence you can use PCR. 330 00:28:54 --> 00:28:58 So the first thing you do is to denature the DNA. 331 00:28:58 --> 00:29:05 And in this case we do that by heat. 332 00:29:05 --> 00:29:10 If you heat up a DNA molecule it will also separate from itself. 333 00:29:10 --> 00:29:16 And we typically heat it up to about 94 degrees. 334 00:29:16 --> 00:29:21 And the consequence of this is this double-stranded piece of DNA 335 00:29:21 --> 00:29:26 molecule will separate into two single-stranded pieces of DNA. 336 00:29:26 --> 00:29:32 And at that point we anneal, hybridize onto the DNA -- 337 00:29:32 --> 00:29:37 I'm not about to do another demonstration here. 338 00:29:37 --> 00:29:43 We anneal onto the DNA oligonucleotides, 339 00:29:43 --> 00:29:48 synthetic little fragments of DNA that are complementary to this known 340 00:29:48 --> 00:29:54 sequence at this end and at this end. So I synthesize a short 341 00:29:54 --> 00:30:00 oligonucleotide which will anneal to this strand at this position. OK? 342 00:30:00 --> 00:30:03 I should have written up the polarities because people often get 343 00:30:03 --> 00:30:07 confused. Here's the 5 prime end of this DNA molecule, 344 00:30:07 --> 00:30:11 the 3 prime end of that DNA molecule, the 3 prime end of that one, 345 00:30:11 --> 00:30:15 5 prime end of that one. So if I'm thinking about the top strand here, 346 00:30:15 --> 00:30:18 the 5 prime end here, the 3 prime end here, and that little 347 00:30:18 --> 00:30:22 oligonucleotide, which in this context we call a 348 00:30:22 --> 00:30:26 primer, would have its 5 prime end here and its 3 prime end here. 349 00:30:26 --> 00:30:30 Thus, the arrow because this is the direction of DNA synthesis. 350 00:30:30 --> 00:30:34 If we're going to synthesize DNA off of this piece of DNA it's going to 351 00:30:34 --> 00:30:38 go in that direction. And then we likewise order up and 352 00:30:38 --> 00:30:42 anneal onto an oligonucleotide that binds to this strand of this 353 00:30:42 --> 00:30:47 sequence. And, again, to show the polarities, 354 00:30:47 --> 00:30:51 here's the 5 prime end. Ah, thank you very much. 355 00:30:51 --> 00:30:55 Here's the 5 prime end, here's the 3 prime end, so that the 356 00:30:55 --> 00:30:59 oligonucleotide primer has its 5 prime end here, its 3 357 00:30:59 --> 00:31:13 prime end here. OK. 358 00:31:13 --> 00:31:18 And now these primers are sitting in such a way, annealed to a template 359 00:31:18 --> 00:31:23 piece of DNA with a 3 prime end facing in this direction, 360 00:31:23 --> 00:31:29 that are ready to be extended by DNA polymerase. 361 00:31:29 --> 00:31:37 Sorry. I'm getting confused about 362 00:31:37 --> 00:31:43 my colors here. You can't see them on the board? 363 00:31:43 --> 00:31:49 You can't see the blue. OK. All right. So the blue, 364 00:31:49 --> 00:31:55 sorry about this. The blue will now be orange. OK? 365 00:31:55 --> 00:32:01 And this one will be pink. OK? So I'm going to extend this 366 00:32:01 --> 00:32:07 pink oligonucleotide with a polymerization reaction to 367 00:32:07 --> 00:32:13 fill in this strand. And I'm going to extend the orange 368 00:32:13 --> 00:32:19 guy likewise to fill in this strand. OK? So this is accomplished by 369 00:32:19 --> 00:32:26 addition of DNA polymerase and the nucleotide precursors that are 370 00:32:26 --> 00:32:33 needed for DNA synthesis, which we abbreviate dNTPs. 371 00:32:33 --> 00:32:37 And if I incubate that in that way I'll get extension from this primer 372 00:32:37 --> 00:32:42 in this direction, extension from this primer in this 373 00:32:42 --> 00:32:46 direction. And what I've done here is to go from one DNA molecule to 374 00:32:46 --> 00:32:51 two DNA molecules. I've duplicated this piece of DNA 375 00:32:51 --> 00:32:56 in a test-tube. The beauty of PCR is that you then 376 00:32:56 --> 00:33:01 go through this process again and again and again and again. 377 00:33:01 --> 00:33:05 And each time you get an exponential increase in the amount of DNA. 378 00:33:05 --> 00:33:09 You go from one to two to four to eight and so on and so on and so on 379 00:33:09 --> 00:33:13 and so on. And in a relatively short time you can now have millions, 380 00:33:13 --> 00:33:18 billions or more copies of your DNA. OK? So that's the basic principle. 381 00:33:18 --> 00:33:22 And, again, we'll go through it in a little more detail in the movie. 382 00:33:22 --> 00:33:26 As I said, the technique was invented by Kary Mullis, 383 00:33:26 --> 00:33:31 an investigator at a company called Cetus. 384 00:33:31 --> 00:33:51 Kary Mullis is an interesting character. I don't have time to 385 00:33:51 --> 00:34:11 tell you the full stories of Kary Mullis. As I told you, 386 00:34:11 --> 00:34:31 he won the Nobel Prize. He's a real California kind of a surfer dude. 387 00:34:31 --> 00:35:05 Very laid back. 388 00:35:05 --> 00:35:09 So, anyway, you can use this technique for lots of applications, 389 00:35:09 --> 00:35:14 as shown here. It's been commercialized in many ways. 390 00:35:14 --> 00:35:18 We now have machines that will go through these various stages of 391 00:35:18 --> 00:35:23 denaturation, annealing and synthesis using a temperature 392 00:35:23 --> 00:35:28 regulated block that will go from 94 degrees to 68 degrees to 72 degrees 393 00:35:28 --> 00:35:32 to allow denaturation, annealing and polymerization around 394 00:35:32 --> 00:35:36 this circle many, many, many times. And if you do this, 395 00:35:36 --> 00:35:39 for example, 30 times, if you go through this cycle 30 396 00:35:39 --> 00:35:43 times you make two to the thirtieth copies of your DNA, 397 00:35:43 --> 00:35:46 which is ten to the ninth DNA molecules, which is a ton. 398 00:35:46 --> 00:35:49 And that can be done in just a couple of hours using a machine such 399 00:35:49 --> 00:35:53 as this. OK. So let me take you through this movie. 400 00:35:53 --> 00:35:56 I'm going to cut it short because the process is gone through 401 00:35:56 --> 00:35:59 in great detail. The narrator is actually Paul 402 00:35:59 --> 00:36:03 Matsudaira who is a professor here at MIT. And Paul really sort of 403 00:36:03 --> 00:37:38 drags it on, but I'm going to -- 404 00:37:38 --> 00:37:41 Basically all he says is what I said, which is you take an individual DNA 405 00:37:41 --> 00:37:45 molecule for which you know sequences, and you run through this 406 00:37:45 --> 00:37:48 reaction multiple times. And each time you do you get a 407 00:37:48 --> 00:37:52 duplication of the DNA sequence. And your book goes through it, too. 408 00:37:52 --> 00:37:56 So I will get this thing posted on the Web. It's not on Monday's quiz 409 00:37:56 --> 00:38:00 so you don't have to worry about that. 410 00:38:00 --> 00:38:04 We can learn about it in the future. Maybe I'll fix it so that on 411 00:38:04 --> 00:38:09 Monday's lecture or Wednesday's lecture, Professor Sive who is 412 00:38:09 --> 00:38:13 giving that lecture can go through it with you. So sorry about this. 413 00:38:13 --> 00:38:18 I also wanted to mention briefly that the enzyme that we use to carry 414 00:38:18 --> 00:38:23 out the polymerization, the particular DNA polymerase that 415 00:38:23 --> 00:38:27 we use is isolated from what we call a thermophilic bacterium isolated 416 00:38:27 --> 00:38:31 from the hot springs. And that's important because we 417 00:38:31 --> 00:38:34 carry out this reaction at a very high temperature. 418 00:38:34 --> 00:38:37 You'll notice that the polymerization was done at 72 419 00:38:37 --> 00:38:41 degrees. Now, your DNA polymerases won't work at 420 00:38:41 --> 00:38:44 72 degrees but this organism grows at very, very high temperatures so 421 00:38:44 --> 00:38:47 its DNA polymerase can function there. And this, 422 00:38:47 --> 00:38:50 again, has turned into a cottage industry. There are hundreds of 423 00:38:50 --> 00:38:53 millions of dollars worth of this particular polymerase called Taq 424 00:38:53 --> 00:38:57 polymerase sold for this application. OK. 425 00:38:57 --> 00:39:02 So let's carry onto the next topic then. So what we've been talking 426 00:39:02 --> 00:39:07 about now is transferring DNA in the case of cloning or amplifying DNA 427 00:39:07 --> 00:39:12 for various applications. For some applications that's not 428 00:39:12 --> 00:39:18 good enough. For certain therapeutic applications, 429 00:39:18 --> 00:39:23 for example, taking human genes and cloning them to make therapeutic 430 00:39:23 --> 00:39:29 proteins or to do gene therapy you cannot do standard cloning. 431 00:39:29 --> 00:39:33 And the reason is that human genes are usually way too big. 432 00:39:33 --> 00:39:37 They have exons and introns, which you learned about, and they 433 00:39:37 --> 00:39:41 can be hundreds of thousands and sometimes millions of nucleotides in 434 00:39:41 --> 00:39:45 length. And that's way too big to fit into one of these plasmid 435 00:39:45 --> 00:39:49 cloning vectors and to efficiently introduce it to bacteria. 436 00:39:49 --> 00:39:53 And so for the purposes of generating clones from human genes 437 00:39:53 --> 00:39:58 we often use a technique called cDNA cloning. 438 00:39:58 --> 00:40:06 And I'll take you through this 439 00:40:06 --> 00:40:13 briefly. Recall that human DNA, human genes are broken up into 440 00:40:13 --> 00:40:19 coding elements that we call exons. And we usually number these exons 441 00:40:19 --> 00:40:26 from left to right, one, two, three. During the process 442 00:40:26 --> 00:40:33 of transcription a copy of this gene is made in the form of a precursor 443 00:40:33 --> 00:40:40 mRNA which has the same sequence. It has both the exons and the 444 00:40:40 --> 00:40:48 introns present. And then before this is translated 445 00:40:48 --> 00:40:56 into protein the information in the introns has to be removed in a 446 00:40:56 --> 00:41:02 process called splicing. And that produces a mRNA which 447 00:41:02 --> 00:41:08 carries the exons lined up next to each other. So the introns are 448 00:41:08 --> 00:41:14 removed, the exons are joined, and it's this mRNA that then gets 449 00:41:14 --> 00:41:20 translated into protein. As I said, this can be very, 450 00:41:20 --> 00:41:26 very large. But this is rather short. And so if we want to make a 451 00:41:26 --> 00:41:32 copy of a gene we can also make a copy of its mRNA. 452 00:41:32 --> 00:41:38 And that might be more efficient and more useful. And this is a 453 00:41:38 --> 00:41:44 technique called cDNA cloning. It was made possible by an 454 00:41:44 --> 00:41:50 invention at MIT, a discovery at MIT of an enzyme that 455 00:41:50 --> 00:41:56 was not known to exist, an enzyme which violated the 456 00:41:56 --> 00:42:02 so-called central dogma. The central dogma being that DNA is 457 00:42:02 --> 00:42:06 transcribed into RNA and that's translated into protein. 458 00:42:06 --> 00:42:11 David Baltimore discovered an enzyme called reverse 459 00:42:11 --> 00:42:19 transcriptase. 460 00:42:19 --> 00:42:24 Which can take the information in RNA and convert it to a DNA form. 461 00:42:24 --> 00:42:29 And he actually won the Nobel Prize for that. So this is the principle. 462 00:42:29 --> 00:42:34 You can take an mRNA which has polarity 5 prime to 3 prime. 463 00:42:34 --> 00:42:39 This mRNA that I produced up there, for example. 464 00:42:39 --> 00:42:44 You can anneal onto it an oligonucleotide primer. 465 00:42:44 --> 00:42:49 This oligonucleotide primer is made of DNA. It has a 5 prime end and a 466 00:42:49 --> 00:42:54 3 prime end. And then you add this enzyme reverse transcriptase, 467 00:42:54 --> 00:43:00 which I'll abbreviate RT. Reverse transcriptase is unique in 468 00:43:00 --> 00:43:06 its ability to copy from an RNA template a DNA strand. 469 00:43:06 --> 00:43:12 And so you'll get a duplex which has RNA on the top and 470 00:43:12 --> 00:43:20 DNA on the bottom. 471 00:43:20 --> 00:43:29 You then get rid of the RNA. 472 00:43:29 --> 00:43:40 You hydrolyze the RNA. 473 00:43:40 --> 00:43:45 So all you're left with is that single strand of DNA. 474 00:43:45 --> 00:43:50 You now anneal on another primer, another oligonucleotide primer which 475 00:43:50 --> 00:43:55 now has a polarity 3 prime to 5 prime in this direction because this 476 00:43:55 --> 00:44:00 one went 5 prime to 3 prime in this direction. And then you 477 00:44:00 --> 00:44:07 add DNA polymerase. 478 00:44:07 --> 00:44:12 Which will extend from this primer and make a full duplex of DNA where 479 00:44:12 --> 00:44:17 both strands are made out of DNA. And this we call a cDNA clone, a 480 00:44:17 --> 00:44:22 copy of the mRNA. cDNA clone. And you can make 481 00:44:22 --> 00:44:27 libraries of cDNA clones from all the RNAs in your cells, 482 00:44:27 --> 00:44:32 for example. And then you can look, 483 00:44:32 --> 00:44:36 using various techniques, at different clones within that 484 00:44:36 --> 00:44:40 library for ones that function in different ways. 485 00:44:40 --> 00:44:44 So I want to take you through a couple of examples of how we use 486 00:44:44 --> 00:44:49 cDNAs in the context of gene therapy. This one comes out of your book. 487 00:44:49 --> 00:44:53 This is an application where there's an enzyme called TPA which 488 00:44:53 --> 00:44:57 will dissolve clots. It's called tissue plasminogen 489 00:44:57 --> 00:45:02 activator. And it's used now to treat people 490 00:45:02 --> 00:45:06 who've had heart attacks or strokes. So what was done was to make a cDNA 491 00:45:06 --> 00:45:10 copy of the TPA gene, introduce that into a plasmid vector 492 00:45:10 --> 00:45:14 with appropriate sequences in red and yellow to allow the expression 493 00:45:14 --> 00:45:18 of that gene in bacteria, transfer it into E. coli by 494 00:45:18 --> 00:45:22 transformation. Now, the E. coli will pump out this 495 00:45:22 --> 00:45:27 enzyme TPA. You can then purify that enzyme and 496 00:45:27 --> 00:45:32 inject it into this happy looking stroke patient to help dissolve the 497 00:45:32 --> 00:45:37 clots that it formed in the formation of the stroke. 498 00:45:37 --> 00:45:41 OK? So that's an example of genetic engineering one of our genes 499 00:45:41 --> 00:45:46 into bacteria to turn them into little protein factories from which 500 00:45:46 --> 00:45:51 you can purify the enzyme and hopefully mitigate the consequences 501 00:45:51 --> 00:45:59 of the disease. 502 00:45:59 --> 00:46:03 A second example, as opposed to putting in a 503 00:46:03 --> 00:46:07 therapeutic protein, you might want to put in a 504 00:46:07 --> 00:46:11 therapeutic gene. So the example here I'll give you 505 00:46:11 --> 00:46:17 is cystic fibrosis. 506 00:46:17 --> 00:46:21 I've mentioned to you in earlier lectures that cystic fibrosis is a 507 00:46:21 --> 00:46:26 genetic disease in which the individuals have a defect in a 508 00:46:26 --> 00:46:31 transporter, an ion transporter, so that in contrast to normal cells, 509 00:46:31 --> 00:46:35 here is a normal cell which has this CFTR transporter which will allow 510 00:46:35 --> 00:46:40 chloride ions to move in and out of the cell, as is a wild 511 00:46:40 --> 00:46:45 type individual. In the case of CF, 512 00:46:45 --> 00:46:50 that transporter is missing. That leads to an inability of the 513 00:46:50 --> 00:46:54 cells to properly regulate their water content, 514 00:46:54 --> 00:46:59 and this leads to disease. So what could you do about this 515 00:46:59 --> 00:47:04 disease? Well, why don't you just put the 516 00:47:04 --> 00:47:09 defective gene back in? We could make a cDNA copy of the 517 00:47:09 --> 00:47:14 cystic fibrosis gene, build it into a vector and introduce 518 00:47:14 --> 00:47:19 it not into bacteria but back into these cells. So we could clone a 519 00:47:19 --> 00:47:24 CFTR cDNA back into these cells. They would, in fact, pick up that 520 00:47:24 --> 00:47:30 DNA and begin to express it. They would make this protein. 521 00:47:30 --> 00:47:34 And that would allow them to transport chloride properly. 522 00:47:34 --> 00:47:39 And they might be “normal”. This actually works extremely well in the 523 00:47:39 --> 00:47:43 tissue culture dish. It doesn't work nearly so well in 524 00:47:43 --> 00:47:48 the context of a human being. Even though we can make viral 525 00:47:48 --> 00:47:53 versions of this, viruses that carry cDNAs, 526 00:47:53 --> 00:47:58 recombinant viruses, and I'll draw that up here. 527 00:47:58 --> 00:48:07 Using the same methods of 528 00:48:07 --> 00:48:12 recombinant DNA technology, I can make a recombinant virus, 529 00:48:12 --> 00:48:17 like a cold virus that now carries a piece of this cDNA for CFTR, 530 00:48:17 --> 00:48:23 and I could use that to infect a person who has CF. 531 00:48:23 --> 00:48:31 That's a person and they have CF so 532 00:48:31 --> 00:48:37 they're not happy. I could introduce into their nasal 533 00:48:37 --> 00:48:43 passage, they could breath in this virus that carries the gene. 534 00:48:43 --> 00:48:48 And if the gene, if the virus infected the cells of the lung, 535 00:48:48 --> 00:48:54 remember the problem occurs in the lung, maybe, if this were very 536 00:48:54 --> 00:49:00 efficient, the person would be happy, we could cure them. 537 00:49:00 --> 00:49:04 The problem is that this introduction of the virus is very 538 00:49:04 --> 00:49:08 inefficient in vivo, in the person. It doesn't work very 539 00:49:08 --> 00:49:12 well. You cannot get enough virus in to infect enough cells to correct 540 00:49:12 --> 00:49:17 the disease in the person. So although gene therapy is very 541 00:49:17 --> 00:49:21 attractive for many diseases such as CF, it actually hasn't worked very 542 00:49:21 --> 00:49:25 well. But there is one example of a cure using gene therapy. 543 00:49:25 --> 00:49:30 And I want to just end by telling you that story. 544 00:49:30 --> 00:49:33 To cure the disease you probably heard about, the disease that causes 545 00:49:33 --> 00:49:36 “the boy in the bubble” syndrome, which is severe combined 546 00:49:36 --> 00:49:39 immunodeficiency, or at least one form of it, 547 00:49:39 --> 00:49:42 these individuals have a defect in an enzyme called ADA, 548 00:49:42 --> 00:49:46 adenosine deaminase. And this results in a very severe 549 00:49:46 --> 00:49:49 immunodeficiency. They don't have their immune cells. 550 00:49:49 --> 00:49:52 They cannot fight infection. So the kids have to stay inside 551 00:49:52 --> 00:49:55 protective chambers to avoid exposure to pathogens, 552 00:49:55 --> 00:49:59 the bubble. For a long time they really weren't well treated. 553 00:49:59 --> 00:50:02 They could be treated with having a little bit of the enzyme. 554 00:50:02 --> 00:50:05 I guess that's not shown here but on the next slide. 555 00:50:05 --> 00:50:09 If you add the enzyme back, even to their blood, you can get 556 00:50:09 --> 00:50:12 some protection. But, nevertheless, 557 00:50:12 --> 00:50:16 the kids did not do terribly well and often died from infections. 558 00:50:16 --> 00:50:19 Here's the disease mechanism. It's caused by a defective enzyme that 559 00:50:19 --> 00:50:23 normally breaks down adenosine. This leads to a buildup of a 560 00:50:23 --> 00:50:26 precursor, or rather a metabolite that kill cells of the immune system, 561 00:50:26 --> 00:50:30 and the individuals have immunocompromised state. 562 00:50:30 --> 00:50:34 If you can just hang on for one or two more minutes. 563 00:50:34 --> 00:50:38 So what's done here, in contrast to the failed example 564 00:50:38 --> 00:50:42 for CF where I said that it's very difficult to get the virus into the 565 00:50:42 --> 00:50:46 cells in the person, what's done in the case of this 566 00:50:46 --> 00:50:51 disease is to take the cells out of the person. And this is called ex 567 00:50:51 --> 00:50:55 vivo gene therapy where you isolate cells from the bone marrow of one of 568 00:50:55 --> 00:50:59 these kids, the stem cells that give rise to the cells of the blood 569 00:50:59 --> 00:51:03 system, and then you infect those cells in vitro with the virus that 570 00:51:03 --> 00:51:07 carries the cDNA of the ADA gene. You isolate those cells that have 571 00:51:07 --> 00:51:11 that gene and then you introduce the cells back into the person. 572 00:51:11 --> 00:51:15 And you can control that process much more carefully, 573 00:51:15 --> 00:51:18 much more efficiently, and you have a much greater 574 00:51:18 --> 00:51:22 concentration of cells that are doing the right thing now. 575 00:51:22 --> 00:51:26 This goes through it in some more detail. It's a version of a slide 576 00:51:26 --> 00:51:30 from your book so you can look at it. 577 00:51:30 --> 00:51:34 It's gene therapy. And I want to point out that 578 00:51:34 --> 00:51:39 although it can work, and it does work, it has one risk, 579 00:51:39 --> 00:51:43 namely that the virus that carries the therapeutic gene actually 580 00:51:43 --> 00:51:48 integrates into the DNA of the cells. And if it integrates into a gene 581 00:51:48 --> 00:51:52 that's important that integration could actually cause a mutation. 582 00:51:52 --> 00:51:57 So there's a risk associated with gene therapy. 583 00:51:57 --> 00:52:00 And, in fact, although this was successful, as you can see here, 584 00:52:00 --> 00:52:04 two kids with SCID, or actually a number of kids with SCID were cured 585 00:52:04 --> 00:52:08 through this ex vivo gene therapy approach, and now they can run 586 00:52:08 --> 00:52:12 around like everyday kids, unfortunately, in this first study 587 00:52:12 --> 00:52:16 of 11 patients, all of whom were “cured”, 588 00:52:16 --> 00:52:20 later two of the kids ended up getting leukemia. 589 00:52:20 --> 00:52:24 And they did because the virus had inserted itself, 590 00:52:24 --> 00:52:28 the genome of the virus had inserted itself next to a gene that when it 591 00:52:28 --> 00:52:32 becomes too active it causes the cells of the immune system to 592 00:52:32 --> 00:52:36 proliferate abnormally leading to leukemia. 593 00:52:36 --> 00:52:40 So although there was a benefit there was also a risk. 594 00:52:40 --> 00:52:45 In this case, families of these kids actually are willing to take 595 00:52:45 --> 00:52:49 that risk because it's such an awful disease. But you have to be aware 596 00:52:49 --> 00:52:54 that in all of these kinds of therapies there's a risk-benefit 597 00:52:54 --> 00:52:57 analysis that has to go on.