Goodbye! The era of individual combat operations in drug mechanism research! ----The whole genome Cas9 library screening principle introduction
Drug research and development is difficult, it is difficult to go to the sky, the mechanism of action research is the key.
On the big screen, Iron Man and the US team are tearing up. The mechanism of anti-tumor drugs is also difficult to distinguish. The various target pathways are on the scene. Which one is strong?
Tyrosine kinase? Angiogenesis? Tumor cell cycle related factors? Histone deacetylase inhibitors? Tumor microenvironment? Cancer stem cells? Abnormal tumor metabolism?
How does the drug mechanism work?
Time is fixed in 2016, are you still knocking genes one by one?
(Chen Bo: Hehehe...)
Wake up! I don’t want to play high-volume in this era, I am embarrassed to go out.
(High-energy warning ahead, cautiously)
The GSK gene's whole-genome Cas9 screening is striking !
The CRISPR/Cas9 vector library was packaged into a lentiviral knockout library containing 122,411 sgRNA targets, of which 19,050 were coding genes.
1864 microRNAs
Overexpression library contains 70,090 SAM-sgRNA targets covering 23,430 coding gene transcripts
The devastating star technology that is about to be hot!
A network to exhaust the target drug related signal pathways to harvest a large number of drug resistance / sensitization related genes preemptive occupation of the target drug research high point to grab high scores article fund application no longer æ„ precision medical good base friend combination drug according to the map 骥 骥 efficacy evaluation tool development artifact
Iron Man, the US captain-style individual combat era has come to an end, the era of the Ark of the Sea of ​​Humanity tactics is coming!
Our slogan is: Simple! rough! effective!
Cas9 library in hand, high score article I have!
First look at the first "Science" in the Cas9 library application. In 2014, Zhang Feng infected the melanoma cell A375 with a knockout library and used velocene (Vemurafenib, a BRAF inhibitor) for drug screening. A series of unreported novel drug-related genes such as NF2, CUL3, TADA2B, and TADA1. At this point, the research on the drug mechanism of Velofini has been fully contracted, and the work of ten years is completed in one year.
In 2015, Zhang Feng used the same routine and re-applied a "Nature". The main difference was that the library was replaced by a knockout library into an overexpression library. The water of the red fruit is ah! Chen Bo has alarmed...
The principle of whole genome Cas9 library screening is not complicated:
- Selected target drugs and cell lines
- Infect cells with Cas9 lentivirus library
- Randomly knock out/overexpress a gene in each cell
- List of sensitizing/resistance-related genes by drug screening and second-generation sequencing
The main experimental procedure for genome-wide Cas9 library screening consists of four phases:
- Pre-experiment - exploring the infection conditions, etc.
- Library screening - high-throughput screening for drug resistance/sensitization genes
- Gene validation - verifying the function of related genes
- Mechanism research - telling stories and improving the grade of the article!
Cas9 library screening is a research mechanism of drug mechanism, but it is not limited to drug research. All the stimulating factors that can affect cell survival and proliferation can study the mechanism behind it: pathogenic virus, hypoxia, oxidative stress, acid-base pressure, temperature, Air pressure, etc. As long as the brain hole is big, CNS is not a dream!
The principle is clear, the method is simple and rude, everything looks easy, is that really true? Chen Bo once again revealed a sinister smile, and listened to the back book, Dr. Professional Pit, how difficult is it to screen the Cas9 library? 》
1.50 Test Strips Per Bottle
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3.10 Parameter Veterinary Urine Test
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[Test Principle]
Urobilinogen:Urobilinogen with diazonium salt produce red violet dyes in strong acid medium.
Bilirubin:The direct bilirubin with dichlorobenzene diazonium produce azo dyes in acid medium.
Ketone:The acetoacetic acid and sodium nitroprusside cause reaction in alkaline medium,which produces red violet compound.
Blood:Hemoglobin acts as peroxidase. It can cause peroxidase release neo-ecotypes oxide (O).(O) oxidizes the indicator and make the color change subsequently.
Protein:It is based on a specific pH indicator attracted by cation on protein molecule,the indicator further ionized and make the color change.This phenomenon is called protein-error-of -indicator principle.
Nitrite:Nitrite and aromatic amine are diazotized to form a diazonium compound.The diazonium compound reacts with tetrahydrobenzo(h)quinolin 3-phenol produce the red azo dye.
Leukocyte:Pyrrole amino acid ester produce free phenol under the hydrolysis of esterase in neutrophile granulocyte,the free phenol couple with phenyl diazonium salt produce purple azo dyes.
Glucose:The glucose oxidized by glucose oxidase catalyzes the formation of glucuronic acid and peroxide hydrogen.Peroxide hydrogen releases neo-ecotypes oxide(O)under the function of peroxidase.(O)oxidizes tetramethyl benzidine, which make the color change.
Specific Gravity:Methyl vinyl ether and maleic acid copolymer is weak acid (-COOH) exchanger,the M+ cation (the major is Na+) in urine reacts with the exchanger and release hydrogen ion,hydrogen ion reacts with indicator produce color change.
pH:The method of pH indicator is applied.
Ascorbic Acid:Ascorbic acid deoxidizes the 2.6-dichlorphenolindophenol dye into colorless in alkaline medium.
Microalbumin:Sulfone phthalein dye has high sensitivity to microalbumin by the method of protein error.
Creatinine:Creatinine with 3,5-Dinitrobenoic acid produce violet compound in alkaline medium.
Calcium:Calcium ion reacts with methyl bromothymol blue produce color change in alkaline medium.
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