1.1 Why we select mouse model:
A mice and human both have similar tumor biology with regard to genome and functional characters, this is the reason why mouse are mostly selected for study of diverse diseases like cancers. Mouse genome is very similar to human, that’s why the basic mechanism of cancer progress and response to its cure are similar.
Different characters at cellular, molecular and anatomical level are similar to humans and have similar functioning in cancer.
1.2 RNA interference- definition:
It is a process which can prevent manifestation of gene at the phase of translation or other way is to obstruct the transcription of exact genes.
RNA intervention is basically a gene-silencing method that subdues the function of objective gene.
1.3. Advantage of this technology:
This experimental proposal offers suppleness by adjusting time and period of gene suppression.
It permits necessary functions or fatal genes to be operated in vivo by having inducible and flexible nature of gene suppression.
Quashing the gene activity is better than to knock out it.
1.4.Use of RNA interference for cure of cancer:
It is widely useful for cancer treatment. Small interfering RNA drugs for cancer has overwhelming advantages over other drugs that are used for cancer cure as well as chemotherapy. Because it acts upon the post translational stage of genic expression that’s why it does not have any harmful effect on DNA. It works by silencing many cancer sponsoring genes.
Fig 1.1 siRNA pathway
2.Mouse model for cancer study using Small interference RNA:
Sequence specific gene silencing is carried out successfully by using small interfering RNAs. It has become an influential instrument for finding of goal and medicines. These are useful especially for cancer treatment. It can inhibit any gene of interest so we can study gene functions and can produce drugs of our demand. This technique has taken the place of knockout mouse models and the production of small molecule because many small molecule inhibitors cause toxic effects in cells.
But it also must be carefully carried out for its successful use in medicines. Enzymes like ribonucleases are used for the breakdown of siRNA which are then passed out by excretion it cannot cross cell membranes because of its high molecular weight, negative charge and water-loving capacity.
For effective delivery systems in cancer research different types of formulations are applied like liposome polymers, or micelles.
For the transfer of small particles to tumors after complete management the physical characters like size, shape, surface charge are important for concern. Long circulating nanoparticles are easily taken up by the tumor vessels because they possess irregular shapes and leaky nature. Once they are taken up the target cells via endocytosis, then they have to release in cytosol. For cancer treatment these steps are very important and should be carried out with great care from the administration site to cytosol in target cells.
Let’s take an example of orthotopic ovarian cancer for this model study, it involves in vivo transfer of siRNA
2.2. Material required for experiment:
a. Commercial reagents:
0.5 M EDTA
Glacial acetic acid
Human ovarian cancer cell lines, Hey A8, SKOVE3, A2780.
Chitosan, low molecular weight
Hank’s balanced salt solution
Taqman miRNA assay.
2×fast syber green master mix
Verso cDNA synthesis kit
Direct-Zol RNA kit
2.3. Equipments required for experiment :
A spectrophotometer for RNA quantification
A thermal cycler
A real time thermal cycler
In vivo imaging system
How to prepare nanoparticle of siRNA/Chitosan:
Chitosan is used for many clinical applications because of its important properties like it is low toxic, biodegradable, biocompatible linear polysaccharide. SiRNA/CH nanoparticles are produced because they have ability to interact with membranes of cells. To transport siRNA into tumors the development of nanoparticle is necessary otherwise siRNA cannot cross cell membranes individually because they have high molecular weights. A process of ionic gelation is carried out for its formation using cationic chitosan and anionic siRNA and TPP.
Following steps are followed for its formation.
In 99.75 ml of water 0.25 of glacial acetic acid is dissolved to produce 0.25 % acetic acid.
Then add CH in 0.25% acetic acid to form a CH solution.
In 100ml of water 0.25 g of TPP is added to produce TPP.
Then to CH solution TPP and siRNA are added with continual mixing to form nanoparticles.
These nanoparticles are then incubated for 40 minutes at four degree Celsius temperature after incubation these particles are centrifuged for 40 min at 4 degree temperature at 14,000 rpm.
Then obtained pellet is washed to eliminate unbound chemicals then these nanoparticles are stored at 4 degree Celsius before using.
How to produce in vivo model of ovarian cancer:
First requirement is of a nude mice its age should be atleast 8-12 weeks mostly available at cancer institutes.
Second requirement is of human ovarian cancer cells like Hey A8 are cultured RPMI1640 fetal bovine serum is provided as the supplementation before they are injected into mice body.
Trypsin enzyme is used for detachment of cells then these are centrifuged at 1200 rpm for five minutes.
Now washed the cells with PBS.
Cells are then resuspended in HBBS.
30g needle is used for injecting these cells into the peritoneal cavity of mice 1×106 per 200 of µl per mouse.
Once the tumor has developed then nanoparticles are inoculated itraperitoneally in the mouse that carries tumor and then 1.25-5 microgram of dose is given per mouse.
How to isolate RNA from tissue, plasma and blood: