がん細胞をターゲットとするノースイースタン大学の生化学研究者、Jiyuan Ahn 氏に会いましょう。
Oh, okay. All right. Hi, my name is June and I’m from uh Penny Boying’s lab and um I study polymerase kappa. Polymerous kappa is um Y family DNA um polymerises that is specialized in the translation synthesis and translation synthesis is a special way of um the DNA um replication system. So there is DNA adducts that can be caused by both internal and external sources that will regularly block the normal replication port and this translation synthesis um the Y family DNA blumerase comes in and um it bypass that adduct but because it is bypassing the DNA damage the adduct it is naturally errorprone um yeah DNA damage um like yeah replication method. So what I study is there’s two main projects in here. Um there are many different um cancer associated single nucleotide polymorphisms found in this polymer scapa and there’s also um region 224 282 on there. Um this region is very inherently flexible and um because it’s flexibility we don’t really get to see the full clear um crystal structure of this region but it’s interesting because they uh that region only exists in um ukarotic cell. So there’s a um yeah procarotic homal but it’s missing that region. So I’m just trying to understand the leading of this region and how these um cancer associated single nucleide polymers are affecting um the cell yeah activity overall. So the previous studies are done in more of the perarotic cell um way. So the protein is purified and then um the thermos shift assay or the primer extension assays are run. But what I would like to do is bring that into more of the cell culture um activity. So I um am I did the PCR trans um causing mutations and then um I uh use the reverse transfaction method to transect all of these um different mutations and in HEC 293 uh T cell line and there’s a wild type hect cell line and then there’s a pool kappa knockout. So all of these variants are uh transfected into the pappa knockout cell line. Yeah. So I treat them with mitocin which is a chemotherapetic drug. Um and then I mainly run two assay um the MTT assay and comet assay. MTTS assay is a cell survival assay and um what it does is that um it reacts with um mitochondrial enzyme in live cells. So um if the cell is live then it forms this um purple crystal and it gets dissolved in the MSO and it’s a color matric assay. The intensity of the purpleeness shows the cell viability and then there’s a comet assay which is interesting assay. Um I um after the treatment I harvest the cell and then I place them in pre-coated microscopic um slide with augur and then um I lice the cell and then I run it um through electrocases and DNA is charged. So more DNA damage um it creates this comet looking yeah form. So the if DNA is not damaged then it remains intact in this circular form and more DNA is damaged it creates this whole collet. Yeah. So it’s a good comparison of this is a wild type um hect cell line after 24 hours of mitoin treatment and then this is without coola knockout cell line. So overall what I found was um it’s a little um interesting because it’s controversial from uh the original 224282 study. So um from the previous study we found that this 224282 loop deleting this loop really did not cause any difference in um the thermal shift and also primer extension assay. So there was really no difference in stability of protein and also primer extension ability. But in here we come assay I see the big difference in um especially with one microgram per milll treatment of mining C uh it’s causing more DNA damage even compared to knockout or also even native of whole gap. So we are kind of thinking this region has something to do with protein interaction with well another with another proteination in a whole bigger like cellular level and then um there’s also another founding in the comet assay as well um we there’s another ones that are kind of aligning with the previous assay um the one G147D and R433R and then this catalytically inactive um mutation kind of follow the same trend as the previous ones. Um it causes more um DNA damage in it. Yeah. So there’s things that I need to look more into um see to see how um they’re making the differences. But the other thing that I need to look more into is that um MTT essay, the sterile survival assay. So when these mutations are made or exposed to this cell line it’s causes a lot of difference on DNA damage in terms of DNA damage but I didn’t see that much difference in terms of cell survival against mitoin and I am not really sure what is exactly like not making it as like yeah what yeah what is not making difference on cell survival But is it causing more DNA damage in there? So that’s uh something that I need to yeah focus more for next study to understand like is it because of the transfection like the yeah efficiency or is it that I’m not treating the cell with enough mining C is it the treatment time or is it really just overall it’s not affecting cell survival rate that much but it’s causing DNA damage. Yeah. So yeah, that’s what I have so far. Yeah. Excellent. So your name again, the lab and the school.
Oh, my name is June. June an excellent good job. Uh
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Here’s a summary of the video titled “Meet Jiyuan Ahn and Biochemistry Researcher at Northeastern University targeting cancer cells”:
June, a researcher from Penny Boying’s lab, studies polymerase kappa, a Y family DNA polymerase involved in DNA replication and damage bypass.
Polymerase kappa specializes in translation synthesis, which allows it to bypass DNA adducts that can block normal replication, although this process is error-prone.
The research focuses on cancer-associated single nucleotide polymorphisms (SNPs) in polymerase kappa and a specific region (224-282) that exists only in eukaryotic cells.
Jiyuan aims to understand how these cancer-associated SNPs affect cellular activity, moving beyond previous studies that primarily focused on prokaryotic cells.
The methodology includes PCR to introduce mutations and reverse transfection to study the effects of these mutations in HEC 293 cells, specifically using a papa knockout cell line.