And there's, besides the genome editing. And people take advantage of this RNAi, for example, to generate this helping RNA, which can interfere in a sequence-specific manner, the function of the gene, by affecting it's translation or transcription. And importantly, as I mentioned, this is most sophisticated techniques that can in a mosaic manner only manipulate subset of cells in the background. And this is can be achieved by a different techniques. But one of the techniques is called or. That is required using the [INAUDIBLE] recombination mass, doing a DNA replication somatic recombination, okay? The mutant allele can be separated. And if one has a ways to mock this mutant gene. And one can achieve the inner daughter cell, the one of the daughter cell containing the mutant. And the other one containing the okay? So again, this takes advantage of this genetically recessive, okay? So in a parental cell, if it's heterozygous, okay? Then it does not have any phenotype, because this notation is recessive, okay? But during the somatic recombination, during DNA replication, and divding, okay? Those somatic recombination, for example, if you can be controlled, be induced by specific recombinants, one can achieve that, the two daughter cells, other things are identical. But the only difference could be that one of the alleles will be achieve the homozygous. And this cell you can study this homozygous mutant phenotype, okay? And the important thing is you need to mark your cells. By for example some frozen marker. So you could easily identify which cell is a mutant cell and which cell is a vertex cell. And again, we are going to talk about briefly later. And only one can achieve the temporal, the T-cell specific medication. One can also use different techniques to control the spatial specificity. Again, one can take advantage of different part of this promoter, that can be both tissue and temporal specific. And this illustrates that example. For example, you can introduce a transgene, and data control of some promoter, okay? And this promoter can determine is spatial tissue specific, or temporal specificity, okay? This is called random insertion. But this random insertion, sometimes this promoter is influenced by the adjacent antigenic modification, okay? So sometimes you might not achieve so high specificity. And people therefore, generally transgenic. What is promoter are flanking by a very, very long, sometimes even more than 100 kb of those endogenons gene. Therefore isolate this promoter or enhancer away from the local environment. And then putting the transgene downstream of this promoter, can recapitulate very high specificity, okay? And there are other ways that, for example, some of my lab are also using is, you can put some specific recombination site in one location of the chromosome. And then every single time, you can introduce the transgene just in that location, okay? And what is the advantage of that, is that if you only change the transgene, make a point rotation. And you always site, you actually integrate in that site. You always have the same expression level, okay? And you can compare in a very good control manner the effect. Again, this is being widely used in fly genetics. And more recently, starting to use in a mass genetics. And more sophisticated way and lower efficient way is just generally the you insert the transgene in the endogenous locus. And what is the transcriptional control? Will be largely intact regulating by the other promoters. And the temporal control, again, can be controlled by a number of different way. For example, if this promoter A is a temporary regulator endogenosy. And then you can couple it with a UAS system, motor A will drive a transcription factor in east to express. And the transcription factor in east, we only recognize this specific sequence, UAS sequence. And therefore, you can use this temporal control promoter A to amplify the signal by generate a lot of transcription factor. And this transcription factor will in turn activate this UAS sequence and its downstream gene to transcribe, okay? And the other way is one can use the chemical control. And similarly using the TTA system, which using the tetracycline or analog which is called doxycycline. And this is a specific transcription factor that will get engineered, from a bacterial transcription factor. Its transcription activity is only, tightly sensitive to the lichen which is doxycycline. And only in the presence of doxycycline, for example, in this case. Only in the presence of doxycycline it combines to the transcription factor to drive gene expression. So this is called [INAUDIBLE] okay? And if you can engineer this transcription factor a little bit. And you can get a reverse reaction, well only in the presence of doxycycline, this transcription will not bind to it. Okay and some subtle confirmation of changes. Similar like our [INAUDIBLE] engineers and synthesis, with some subtle changes, you can have both on and off response to the somatic, okay? And likewise, if you change this transcription factor to the Cre recombinase, then you are generating using a Cre recombinase, for example, to out of. And then you can allow this gene to express. You can also achieve the temporal and spatial recognition. And other ways is, one can also using the virus. And other for example, electro provision masses to express DNA, again, introduced in a specific cells. And again this is a powerful, the reason is the nervous system is so complicated. So one need to develop this complicated genetic techniques to perturb, both spatially and temporary, the gene's function to achieve perfect control to understand the specific action, okay? If you don't have this spatial and temporal control, then likely the might be nonspecific. Or there will be some developmental compensation, okay? And to achieve this specific perturbation, one can also using this so-called intercessional laboring to control subset of the neurons. Again, this can be used in different combination to only intercept, for example, the cell, both labeled by promoter A and B. You can use the interception to label what, okay? How do you do it in this way? What you can put this promoter here. And this promoter will drive this UAS to express. However because this is stock. So even if the GAL4 expressed, this transgene will not get expressed, because at the beginning of this transgene there is a okay?. A stock set in front, well only when is expressed, because p will try this to express. And this will recombine this FRT site together to eliminate this stock set. And then this transgene can get expressed. And one can also use other combination. For example, the GAL80 is a specific protein that can bind to GAL4 to inactivate GAL4, okay? Again, this GAL80, is a transcription repressor borrowed from yeast, only recognized GAL4, okay? So in this case, if you have this promoter A expressed, then the GAL4 will express to drive this gene to express. However, if the inner cell expressed, promoter B, the Kl 80 will get express and this Kl 80 will actively inhibit the Kl 4. So the total gene expressed will only be part of the promoter A, okay? So again people can design different logic gate, to intercept, to control the expression of the transgene at different T cells in a spatial and temporal manner. And this allowed one to fine tune which cell that you're affecting. And that's what I used to study the nervous system, which has so many cell and different function, okay? And then one can take advantage of the observing part, which is for example, using the to looking at which gene get expressed at which brain region. For example, this is showing one of the gene only expressed in a special in a brain, and not in other region, okay? And more skillably, people can use the gene chip to study many genes at the same time. And these days the second generation, the next generation sequencing, actually allow more and more people to use, for example, just seek to start a wish gene express in which is. And this will allow one to identify to the diseased gene, for example. And this illustrates, how the genome sequencing revolutionize our study of the disease. And how many the gene's sequencing power? How many output per instrument that grows over time, okay? And you can see there's a sharp increase. And the price on the other hand has dropped in an exponential manner.
