Im working on a genetics science project for my school and I need help with some definitions that my mentor at VCU included in her email for the procedure:
>COS-7 Cells
>DMEM
>Penicillin/ Streptomycin
>Fetal Bovine Serum
>Lipofectamine (protocol)
>Vectashield
>DAPI (microscope stain)
>FITCI (microscope stain)
>transfection efficiency
I am using a gene called RAI1 and seeing how it reacts when injected into COS-7 cells. The mutation and deletion of this gene results in Smith-Magenis Syndrome, but it's very rare.
The following reply is by : Shane M :
COS-7 Cells:
COS is a cell line often used by biologists when studying the monkey virus SV40. It is also often used to transfect cells to produce recombinant proteins for molecular biology, biochemistry, and cell biology experiments. Two forms of COS cell lines commonly used are COS-1 and COS-7.
The COS cell line was obtained by immortalizing a CV-1[1] cell line derived from kidney cells of the African green monkey with a version of the SV40 genome that can produce large T antigen but has a defect in genomic replication.[2]
When an expression construct with an SV40 promoter is introduced into COS cells, the vector can be replicated substantially by the large T antigen.
The word COS is an acronym, derived from the cells being CV-1 (simian) in Origin, and carrying the SV40 genetic material.
DMEM:
Dulbecco’s modified essential medium (DMEM) is a growth media commonly used in mammalian tissue culture experiments.[1] It is a derivative of Eagle's minimal essential medium A few important variances within the medium are the inclusion or concentration of the following compounds: Phenol Red, Glucose, and GlutaMAX.
Penicillin:
Penicillin (sometimes abbreviated PCN or pen) is a group of Beta-lactam antibiotics used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms.
“Penicillin” is also the informal name of a specific member of the penicillin group Penam Skeleton, which has the molecular formula R-C9H11N2O4S, where R is a variable side chain.
Streptomycin:
treptomycin is an antibiotic drug, the first of a class of drugs called aminoglycosides to be discovered, and was the first antibiotic remedy for tuberculosis. It is derived from the actinobacterium Streptomyces griseus. Streptomycin is a bactericidal antibiotic[3]. It kills sensitive microbes by inhibiting protein synthesis; more specifically, it binds to the 16S rRNA of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This prevents initiation of protein synthesis and leads to death of microbial cells. Humans have structurally different ribosomes from bacteria, thereby allowing the selectivity of this antibiotic for bacteria. Streptomycin cannot be given orally, but must be administered by regular intramuscular injection. An adverse effect of this medicine is ototoxicity, which can lead to temporary hearing loss.
Fetal Bovine Serum:
Fetal bovine serum (or fetal calf serum) is serum taken from the fetuses of cattle. Fetal bovine serum (FBS) is the most widely used serum in the culturing of eukaryotic cells.
The globular protein, bovine serum albumin (BSA), is a major component of fetal bovine serum. The rich variety of proteins in fetal bovine serum maintains cultured cells in a medium in which they can survive, grow, and divide. However, the presence of the many proteins in fetal bovine serum can also mean that purification of secreted proteins from the cells can be a more laborious process due to having to remove the contaminating proteins during the purification process.
Fetal bovine serum is commercially available from many manufacturers, and because the cells are highly sensitive, it may be wise to take time to adapt the cells when changing fetal bovine serum from different manufacturers, such as by mixing 50% of the old serum with 50% of the new serum, and leaving the cells in that environment for a while.
Fetal bovine serum should be stored frozen before adding to media in order to prevent contamination. When thawing fetal bovine serum, even though it will take longer, it should be warmed at room temperature and not in a 37-degree water bath. When performing cell culture, the bottle of fetal bovine serum should be opened and closed in a biosafety cell culture hood, and in-person training should be obtained on how to perform sterile manipulations.
Lipofectamine (protocol):
Materials:
bullet Lipofectamine (Invitrogen)
bullet IMDM containing 10% fetal bovine serum, 1% glutamine, 1% aa
bullet IMDM containing 1% glutamine
bullet IMDM containing 20% fetal bovine serum, 1% glutamine, 1% aa
1. In a six-well or 35 mm tissue culture plate, seed ~2x 105 cells per well in 2 ml IMDM containing 10% FBS and nonessential amino acids.
2. Incubate the cells at 37°C in a CO2 incubator until the cells are 70-80% confluent. This will usually take 18-24 h.
3. Prepare the following solutions in 12 x 75 mm sterile tubes:
Solution A: For each transfection, dilute 2 μg DNA (plasmid) in 375 μl serum-free IMDM (containing nonessential amino acids).
Solution B: For each transfection, dilute 12 μl LIPOFECTAMINE Reagent in 375 μl serum-free IMDM.
4. Combine the two solutions, mix gently, and incubate at room temperature for 15-45 min. The solution may appear cloudy, however this will not impede the transfection.Wash the cells once with 2 ml serum-free IMDM.
5. For each transfection, add 750 μl serum
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