Applications of recombinant DNA technology

Question 1

traits of the Asilomar conference

Answer 1

~ use bacterial hosts that cannot survive in natural environment
~ use vectors that can survive in specified hosts

Question 2

examples of monoclonal antibodies

Answer 2

such as infliximab and rituximab

Question 3

advantages and disadvantages of recombinant protein in bacteria

Answer 3

Escherichia coli
Bacillus subtilis
Streptomyces spp. ▲Potentially very high expression levels ▲Inexpensive and simple to culture Streptomyces spp. ▲Fast growing
▲Easily scalable ▲Simple transformation protocols
▲Many parameters can be altered to optimize expression
Disadvantages ◉Limited post-translational modifications (glycosylation, proteolytic maturation, Inefficient disulfide bond formation) ◉Limited protein size and complexity ◉Usually require protein extraction and refolding ◉Codon usage different to eukaryotes ◉Contaminants(traces of endotoxins in Gram-)

Question 4

expression in recombinant protein in yeast

Answer 4

Yeast
Saccharomyces cerevisiae
Pichia pastoris
▲Post-translational modifications ▲Secretion Pichiapastoris ▲Fast growing Etc. ▲Inexpensive to culture
▲Robust cellular structure
▲Synthesis of complex and multisubunitproteins
Disadvantages ◉Glysosylationpattern dissimilar to humans ◉Complex scaling up fermentation

Question 5

expression of recombinant protein in mammalian cells

Answer 5

Mammalian cells
CHO
Murine cell lines
Monkey cell lines
▲Efficient protein folding
▲Innate secretion mechanisms
▲Large proteins can be expressed ▲Synthesis of complex and multi subunit proteins Human cell lines
▲Greatest post-translational modification efficiency ▲Endotoxin-free
Disadvantages ◉Expensive culture media
◉Slow growing ◉Complex growth requirements ◉Sensitive to osmotic shock and shear stress ◉Contaminants(can harbor human pathogens)

Question 6

what are disadvantages of diabetes therapy in cows and pigs

Answer 6

slight structural differences, developmental of antibodies, large number of animals were killed

Question 7

pre pro insulin

Answer 7

The primary structure of insulin is formed by 4 regions: the signal peptide and the regions called B, C, and A

Question 8

pro insulin

Answer 8

The first proteolytic cleavage removes the signal peptide. This cut is made by the signal peptidase within the endoplasmic reticulum, there the protein also gets the two disulphide bonds that characterize its tertiary structure.

Question 9

active insulin

Answer 9

is produced by two additional proteolytic cleavages, which cause the removal of the C region (this happens in the secretory granules of pancreatic beta-cells).

Question 10

tissue plasminogen activator is used to treat

Answer 10

myocardial infarction

Question 11

recombinant vaccine

Answer 11

is a vaccine produced through recombinant DNA technology, This involves inserting the DNA encoding an antigen(such as a bacterial surface protein) that stimulates an immune response into bacterial or mammalian cells, expressing the antigen in these cells and then purifying it from them

Question 12

gene therapy

Answer 12

is a method of curing genetic disorders by introducing functioning genes into a patients body

Question 13

what are the 2 kinds of gene therapy

Answer 13

in vivo and ex vivo

Question 14

somatic gene therapy

Answer 14

treats patients by introducing the genes to body cells

Question 15

germ line gene therapy

Answer 15

targets egg and sperm cells its currently impossible

Question 16

what are the 4 problems with gene therapy

Answer 16

~short lived nature of cure
~immune response
~problems with viral vectors
~multigene disorders

Question 17

there are 2 types of transgenes

Answer 17

~integrated : are stable and could give a cure, are random insertions in heterochromatin and can be inactivated, in euchromatin can disrupt host genes
~not integrated: for episomes (plasmids) random mutagenesis is not an issue, epression is transient, repeated treatments are necessary

Question 18

the most common viral vectors are

Answer 18

retroviruses, adenoviruses, adeno associated viruses, herpes

Question 19

retroviral vectors

Answer 19

can only infect dividing cells only as they cannot penetrate the nuclear membrane

Question 20

lentiviruses

Answer 20

are retroviruses that can infect both dividing and non dividing cells

Question 21

adenoviruses

Answer 21

are non enveloped viruses, containing dsDNA, can infect both dividing and non dividing cells.
But they cannot integrate with the host genome

Question 22

Adeno associated viruses

Answer 22

do not cause inflammation, do not make antibodies against themselves, can enter non dividing cells, they integrate successfully into one spot in the genome