Genetic Manipulation I Flashcards
What are the major applications of plasmids?
- Expression of recombinant protein in different host cells, e.g. production of proteins
- Expression of mutant proteins in different host cells
- Manipulation of genetic expression
- Expression and study of the regulatory sequences (Promoter) of gene expression
Describe Application #1-Recombinant protein production
- Recombinant protein is a manipulated form of a protein encoded by a gene (recombinant DNA where the gene coding sequence is cloned in a system that supports expression of the gene and translation of messenger RNA).
- The recombinant protein can be in the wild type form or in a mutated form.
What is Posttranslational modification?
The covalent and generally enzymatic modification of proteins following protein biosynthesis.
Postranslational modification: Hydroxylation
Attaches a hydroxyl group (-OH) to a side chain of a protein
Postranslational modification: Methylation
Adds a methyl group, usually a lysine group or arginine residues
Postranslational modification: Lipidation
Attaches a lipid, such as a fatty acid, to a protein chain
Postranslational modification: Acetylation
Adds an acetyl group to an N-terminus of a protein or at lysine residues
Postranslational modification: Disulfide bonds
Covalently links the S atoms of two different cysteine residues
Postranslational modification: SUMOylation
Adds a small protein SUMO (small ubiquitin-like modifier) to a target protein
Postranslational modification: Ubiquitination
Adds ubiquitin to lysine residue of a target protein for degradation
Postranslational modification: Glycosylation
Attaches a sugar, usually to an N or O in an amino acid side chain
Postranslational modification: Phosphoyrlation
Adds a phosphate to serine, threonine or tyrosine
What are the pros of bacterial expression systems?
They require simple culture conditions (e.g. media and additives), which are scalable and incurs low cost
What are the cons of bacerial expression systems?
- Difficulty in expressing some mammalian proteins
- Protease contamination from host cells leading to degradation of expressed protein
- Endotoxin contamination
- Lack of post-translational modification
What are the pros of yeast expression systems?
- Well-defined, economic eukaryotic expression system
- Suitable for expression of secretory proteins as well as intracellular proteins
- High protein yield, lesser expression time, post- translational modifications and requires simple media
What are the cons of yeast expression systems
- Hyperglycosylation of proteins, unlike mammalian system, it offers both N and O linked oligosaccharides for glycosylated proteins
- Fermentation is necessary for high protein yield
What are the pros of insect cell expression systems?
- Most used technique to express mammalian proteins, which require post-translational modifications
- Most similar to mammalian expression systems
- Can be used both in adherent and suspension cultures
- Purification process is easy
- Baculoviruses are safe to work with compared to mammalian viruses
What are the cons of insect cell expression systems?
- Time consuming cloning procedure of desired gene in baculovirus vector
- Requiring very expensive media
- Glycosylation is different from that of mammalian system resulting in improper maintenance of epitopes on protein
What are the pros for mammalian expression systems?
- An ideal choice for the production of therapeutic proteins and vaccines
- Both plasmid or virus based vectors can be used
- Offering desired post-translational modifications and proper protein folding
What are the cons of mammalian expression systems?
- High cost of protein production, because of slow cell growth, expensive media and culture conditions (continuous CO2 supply, expensive transfection reagents)
- High protein yield can be obtained only by suspension cultures. e.g. Chinese Hamster Ovarian (CHO) cells and Human Embryonic Kidney (HEK 293) cells
What are the pros of cell free expression systems?
- Expressing proteins in a cell free environment using cell extract, DNA template, amino acids and cofactors
- A simple process, protein expression and purification can be done in a short period of time (1-2 days)
What are the cons of cell free expression systems?
- Degradation of protein by exogenous proteases from cell extracts
- The extracts are expensive
- Small yields
What are the considerations for recombinant protein rpdocution?
- Yield
- Costs
- Protein of interest, e.g. intracellular or extracellular protein, post-translational modification etc.
- Culture condition
- Contamination, e.g. endotoxin etc.
- Protein degradation
What is genetic manipulation?
- The process of inducing changes in gene expression and the expression of novel genes.
- Manipulation of gene expression allows us to study the impact of proteins on genes or other proteins.
Describe the control of gene expression in mammalian cells
Gene expression can also be regulated at the promoter of the gene through outside in regulators, e.g. growth factors and cytokines.
Define transformation
A process of horizontal gene transfer by which some bacteria or non-animal eukaryotic cells take up foreign non-viral genetic material (naked DNA)
What is transfection?
A process of deliberately introducing naked or purified nucleic acids into eukaryotic cells normally by using a plasmid vector
Define transduction
A process by which foreign DNA is introduced into a cell by virus or viral vector
What are Small interfering RNAs (siRNAs)?
dsRNAs that activate RNAi, leading to the degradation of mRNAs in a sequence-specific manner dependent upon complementary binding of the target mRNA
What are Short-hairpin RNAs (shRNAs)?
Contains a loop structure that is processed to siRNA and also leads to the degradation of mRNAs in a sequence-specific manner dependent upon complementary binding of the target mRNA
siRNA v shRNA
siRNA:
- siRNA only functions in the cytosol of target cells
- Once introduced into target cells, the siRNA needs to be separated (single stranded) then it can bind in a sequence specific order to the traget mRNA
- Only one strand of siRNA can bind to the target mRNA and leads to its degradation
- Transient function (siRNA reduces as cells proliferate)
shRNA:
- Introduced into the cell via a vector
- shRNA is continually expressed by the target cell
- shRNA can go into the cytosol to and be processed into siRNA
- Long lasting effects, plasmid shRNA can be passed on from parental cells to daughter cells
RNA interference (RNAi
What are miRNAs (miRNAs)?
Single stranded RNAs of about 20–24 nucleotides. This kind of RNAs act as endogenous post-transcriptional repressors to downregulate gene expression
Compare the function of DNA to RNA?
- DNA replicates and stores genetic information. It is a blueprint for all genetic information contained within an organism.
- RNA converts the genetic information contained within DNA to a format used to build proteins, and then moves it to ribosomal protein factories. RNA has other structural, regulatory and catalytic functions, e.g. rRNAs & microRNAs.
What are specific miRNAs that are currently being pursued as clinical candidates?
Explain CRISPR-Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR Associated protein 9 (Cas 9)
- DNA invasion- Foreign DNA from a virus or plasmid invades the cell
- Invading DNA is incorporated into CRISPR arrary-DNA fragments from the invading DNA are incorporated into the CRISPR locusd as spacers. The exact mechanism of incorporation remains unknown
- Pre-crRNA transcription- The cell constituitvely transcribed a repeat/spacer group into pre-crRNA. Black boxes represent repeats. Grey boxes represents spacers. The red box represents the spacer corresponsing to the invading DNA
- Guide RNA formation- Constitutively expresses transactivating RNA (tracrRNA) base pairs with the CRIPR repeat sequences in the pre-crRNA. RNase III, Csn 1, and other unidentified CRISPR associated proteins modify the pre-crRNA/tracrRNA duplex form a guide RNA
- Cas9 Activation- Inactive Cas9 protein binds to the RNA and becomes activated
- Target Binding- The activated guide RNA/Cas9 complex binds with the target DNA. The localization occurs stochastically
Current progress in CRISPR/Cas9
Over-expression of target gene
How do you measure the efficiency of genetic manipulation?
Direct read out on target protein expression
- Western blotting
- Immunofluorescence stainining
Changes in the phenotypes
Showing characteristics of the target protein or vector
- Resistant to antibiotics
- Expression of tag protein
