Genetic Manipulation I

Question 1

What are the major applications of plasmids?

Answer 1

• 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

Question 2

Describe Application #1-Recombinant protein production

Answer 2

• 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.

Question 3

What is Posttranslational modification?

Answer 3

The covalent and generally enzymatic modification of proteins following protein biosynthesis.

Question 4

Postranslational modification: Hydroxylation

Answer 4

Attaches a hydroxyl group (-OH) to a side chain of a protein

Question 5

Postranslational modification: Methylation

Answer 5

Adds a methyl group, usually a lysine group or arginine residues

Question 6

Postranslational modification: Lipidation

Answer 6

Attaches a lipid, such as a fatty acid, to a protein chain

Question 7

Postranslational modification: Acetylation

Answer 7

Adds an acetyl group to an N-terminus of a protein or at lysine residues

Question 8

Postranslational modification: Disulfide bonds

Answer 8

Covalently links the S atoms of two different cysteine residues

Question 9

Postranslational modification: SUMOylation

Answer 9

Adds a small protein SUMO (small ubiquitin-like modifier) to a target protein

Question 10

Postranslational modification: Ubiquitination

Answer 10

Adds ubiquitin to lysine residue of a target protein for degradation

Question 11

Postranslational modification: Glycosylation

Answer 11

Attaches a sugar, usually to an N or O in an amino acid side chain

Question 12

Postranslational modification: Phosphoyrlation

Answer 12

Adds a phosphate to serine, threonine or tyrosine

Question 13

What are the pros of bacterial expression systems?

Answer 13

They require simple culture conditions (e.g. media and additives), which are scalable and incurs low cost

Question 14

What are the cons of bacerial expression systems?

Answer 14

• Difficulty in expressing some mammalian proteins
• Protease contamination from host cells leading to degradation of expressed protein
• Endotoxin contamination
• Lack of post-translational modification

Question 15

What are the pros of yeast expression systems?

Answer 15

• 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

Question 16

What are the cons of yeast expression systems

Answer 16

• Hyperglycosylation of proteins, unlike mammalian system, it offers both N and O linked oligosaccharides for glycosylated proteins
• Fermentation is necessary for high protein yield

Question 17

What are the pros of insect cell expression systems?

Answer 17

• 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

Question 18

What are the cons of insect cell expression systems?

Answer 18

• 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

Question 19

What are the pros for mammalian expression systems?

Answer 19

• 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

Question 20

What are the cons of mammalian expression systems?

Answer 20

• 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

Question 21

What are the pros of cell free expression systems?

Answer 21

• 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)

Question 22

What are the cons of cell free expression systems?

Answer 22

• Degradation of protein by exogenous proteases from cell extracts
• The extracts are expensive
• Small yields

Question 23

What are the considerations for recombinant protein rpdocution?

Answer 23

• Yield
• Costs
• Protein of interest, e.g. intracellular or extracellular protein, post-translational modification etc.
• Culture condition
• Contamination, e.g. endotoxin etc.
• Protein degradation

Question 24

What is genetic manipulation?

Answer 24

• 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.

Question 25

Describe the control of gene expression in mammalian cells

Answer 25

Gene expression can also be regulated at the promoter of the gene through outside in regulators, e.g. growth factors and cytokines.

Question 26

Define transformation

Answer 26

A process of horizontal gene transfer by which some bacteria or non-animal eukaryotic cells take up foreign non-viral genetic material (naked DNA)

Question 27

What is transfection?

Answer 27

A process of deliberately introducing naked or purified nucleic acids into eukaryotic cells normally by using a plasmid vector

Question 28

Define transduction

Answer 28

A process by which foreign DNA is introduced into a cell by virus or viral vector

Question 29

What are Small interfering RNAs (siRNAs)?

Answer 29

dsRNAs that activate RNAi, leading to the degradation of mRNAs in a sequence-specific manner dependent upon complementary binding of the target mRNA

Question 30

What are Short-hairpin RNAs (shRNAs)?

Answer 30

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

Question 31

siRNA v shRNA

Answer 31

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

Question 32

RNA interference (RNAi

Answer 32

Question 33

What are miRNAs (miRNAs)?

Answer 33

Single stranded RNAs of about 20–24 nucleotides. This kind of RNAs act as endogenous post-transcriptional repressors to downregulate gene expression

Question 34

Compare the function of DNA to RNA?

Answer 34

• 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.

Question 35

What are specific miRNAs that are currently being pursued as clinical candidates?

Question 36

Explain CRISPR-Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR Associated protein 9 (Cas 9)

Answer 36

1. DNA invasion- Foreign DNA from a virus or plasmid invades the cell
2. 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
3. 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
4. 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
5. Cas9 Activation- Inactive Cas9 protein binds to the RNA and becomes activated
6. Target Binding- The activated guide RNA/Cas9 complex binds with the target DNA. The localization occurs stochastically

Question 37

Current progress in CRISPR/Cas9

Answer 37

Question 38

Over-expression of target gene

Answer 38

Question 39

How do you measure the efficiency of genetic manipulation?

Answer 39

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