Dispen

On 11/17/2019 at 3:35 PM, CharonY said:

Unfortunately it is often difficult to predict how different cell lines react to certain chemicals. There are certain cell lines which are considered more hardy, and often e.g. primary cell lines are more sensitive than many carcinomas. HepG2 is often used as it is a bit less resilient (and probably less full of viruses) compared to e.g. HeLa, but there are folks who prefer to use primary cell lines, as the results are probably closer in vivo conditions. But there is no simply way to extrapolate those values (to my knowledge) and most data is obtained empirically.

In other words, HepG2 data can not be easily translated to other cell lines. Using whole animals things are getting more complicated. Remember, the dose you use directly at the cell is independent on the animal (as we work on the cellular level). However, during in vivo experiments you will need to account for the interaction of the chemical with the body (ADME- absorption, distributon, metabolism, excretion). I.e. if you apply the chemical to a rat, depending on how you apply it (e.g. orally, intravenously), the bioavailability of the chemical and how it is metabolized will determine how much will actually end up in the liver to do damage. Moreover, metabolization can inactive it (e.g. making it more hydrophilic and eliminate from the body quicker than the original compound) or conversely make it more toxic. There is a reason why in many cases we still rely on animal data, rather than solely on cell lines to assess human toxicity.

Thanks a lot !

In my experiment, I use a HepG2 cell line for a cytotoxicity assay with Alamar Blue dye, to test toxicities of different metal ions on the liver cells. HepG2 is a liver carcinoma cell line from a Homo Sapiens (human) origin. I know that usually liver cell is used for cytotoxicity assay, however, I wonder what will happen if I use brain cell line instead? And what if I use rat cells instead of human cells? I noticed a study comparing a mouse neuroblastoma line (neuro-2a) to primary neurons found that neuro-2a cells were much less sensitive to neurotoxins, possibly due to a lack of important membrane receptors and ion channels. It means that some neuronal cell lines produce undesirable unrepresentative results. Also, neuronal cells are usually used for investigation of neurotoxicity but not toxicity to human via normal routes of toxic substance intake and degradation, i.e. detoxification in liver. Nonetheless, does it mean the results produced by neuronal cell line will be very different compared with by HepG2? 

If I use rats instead of human liver cell line, I think the results will be similar but the dose used to produce the same response level may be lower because rats are much smaller in size than human? Am I right?

13 hours ago, BabcockHall said:

Ninfa, Ballou, and Benore's textbook on the biochemistry and biotechnology laboratory has a good introduction to this topic.

Thanks for the information. I will take a look.

I have to submit my assignment on time and I need some help. Could someone provide some help or source of reference to me? Thanks.

1.His-tag is well known for its affinity towards nickel and other metal ions. However, other metal-binding proteins present in the cell lysate can also be eluted with our target protein using the present purification protocol (using metal chelating column). How to modify the elution step to improve the purity of the His-tagged fluorescent protein?  

2.Affinity tag may interfere the native function of the target protein. Using recombinant DNA techniques, I can introduce a protease cleavage site into the construct so that the affinity tag can be removable. What are the criteria of selecting the protease to remove the tag? How to design a recombinant DNA construct encoding a removable His-tag fused with a fluorescent protein?

3.The presence of the fluorescent protein was detected by blue light illumination to track the enrichment and purification process of fluorescent protein. What is the advantage of this detection method? And how can we examine the purity of the eluted protein?

9 hours ago, Vexen said:

When cell encounter dsDNA they presume it viral based and degrade it through RNAi. The Dicer enzyme cleaves short hairpin RNAs (shRNAs) into siRNA (single stranded RNA). The RISC complex binds the siRNA and guide it to complementary mRNA strand where it gets cleaved and eventually degraded. This is how cells degrades viral dsRNA.

RNAi will still work for multiple viruses invading a single host cell since multiple dicer proteins recognizes dsDNA.

^ Lee, Young Sik; Nakahara, Kenji; Pham, John W; Kim, Kevin; He, Zhengying; Sontheimer, Erik J; Carthew, Richard W (April 2004). "Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways". Cell . 117 (1): 69–81

Thanks a lot for your explanation! Very helpful!

If two viruses with totally different dsRNA invade a same host cell, will RNAi still work?