BIOCH Y1 S1: Gene Technologies Flashcards
1
Q
where is HGH secreted and what is its function?
A
- pituitary gland
- regulates growth and development
2
Q
how is HGH used in HRT?
A
- to treat Pts w/ hypopituitarism, achondroplasia or dwarfism
- must treat early while bone growth is still possible
3
Q
disadvantages of using HGH in HRT and how were these overcome?
A
- expensive: HGH derived from cadavers, need 80 cadavers per year for 8-10 years
- significant risks e.g. Creutzfeldt-jakob
- we can now produce HGH in bacteria (like insulin)
4
Q
recombinant plasmid steps
A
- vector (bacterial plasmid) includes sequences for transcription and translation in target cell
- foreign gene and plasmid are cut at specific recognition sites using endonucleases
- foreign gene ligated into plasmid
- plasmid inserted into E. coli which then expresses the foreign gene
- antibiotic
5
Q
4 components needed for PCR
A
- template DNA
- DNA polymerase in a compatible buffer
- primers
- dNTPs
6
Q
issue in PCR and how is this addressed
A
- to separate the 2 strands we need to heat however this will denature DNA polymerase and prevent primers from annealing
- so we use a thermostable DNA Taq polymerase
7
Q
PCR steps
A
- denaturing: DNA heated to 90 deg separate strands by breaking H bonds
- annealing of primers: cool DNA to 60 deg allow primers to anneal and H bonds to reform
- elongation: heat DNA again to 72 deg allow DNA Taq polymerase to attach and copy each single strand from 5’-3’ using free nucleotides
8
Q
purpose of antibiotic resistance gene
A
- to test that bacteria have uptaken the plasmid they are treated w/ an antibiotic
- only those that survive will have taken up the plasmid and therefore be able to express the target gene
9
Q
when to use mammalian/insect cells vs yeast/bacteria
A
- bacteria: quick and cheap so used for simple proteins however lack of post-translational modifications
- insect: post-translational modifications for prokaryotic or simple eukaryotic proteins
- mammalian: if post-translational modifications need to be made for
complex eukaryotic proteins - yeast: greatest yield of proteins
10
Q
therapeutic cloning vs reproductive cloning
A
- therapeutic cloning: derive cell lines w/ same genome as nuclear donor (can use SCNT to get embryo > embryonic stem cells)
- reproductive cloning: to produce a person/animal (uses SCNT to get embryo > implanted into surrogate)
11
Q
2 methods of therapeutic cloning
A
- SCNT > embryonic stem cells
- induced pluripotent stem cells (iPSCs)
12
Q
diff levels of cell potency
A
- totipotent: can form a full organism
- pluripotent: can differentiate into any somatic cell
- multipotent: can only give rise to cell types within their lineage
13
Q
process of SCNT
A
- donor somatic nucleus implanted into enucleated egg
- forms zygote which divides and develops into a blastocyst (early embryo)
- collect and culture embryonic stem cells from embryo
- embryonic stem cell lines can be induced to form diff types of specialised cells
14
Q
why do we use therapeutic cloning
A
- treat diseases by replacing damaged tissue
- prevent immunological tissue rejection
- reduce wait times for organ transplants
15
Q
why can SCNT be considered unethical and what are some alternatives
A
- embryo gets destroyed > when is the beginning of life?
- instead use iPSCs > reprogram adult cells to be pluripotent
16
Q
how are induced pluripotent stem cells (iPSCs) produced
A
- skin cells removed from Pt and cultured
- vector carrying a gene is added to the cells which are cultured again
- cells grow to pluripotent cells > induced to be specialised
17
Q
challenges of using iPSCs
A
- telomeres not reset to embryo-like state > more aging
- teratoma (unwanted tissue) formation
- use of viral vectors can lead to cancer-causing genes and fewer tumour-suppressing genes e.g. Rb/p53
18
Q
limitations of gene therapy (gene addition w/ viral vector)
A
- not as efficient b/c use of vectors means not all cells will take it up > have to culture lots of cells
- incorrect placement of new gene may cause cancer
- viral vectors can stimulate immune response > rejection
- manufacturing problems/cost
19
Q
advantage of gene editing over gene therapy (addition)
A
- more precise correction of endogenous gene means less risk of cancer
- repaired gene will still be under control of natural promoter > cell will make correct amount of protein
20
Q
origin of CRISPR-Cas9
A
- bacterial immune system
- store viral DNA as spacers
- when the same virus attacks, spacer RNA binds to gRNA to form chimeric (joined) RNA
- Cas9 (endonuclease) binds to chimeric RNA which guides it to the PAM sequence to cut viral DNA
21
Q
how CRISPR works in the lab
A
- complementary sgRNA is synthesised artificially
- sgRNA introduced into the cell w/ target DNA
- Cas9 (endonuclease) binds to sgRNA which guides it to the PAM sequence to cut DNA
- cell repairs DNA and introduces a mutation
22
Q
clinical challenges of gene editing using CRISPR
A
- viral vector could lead to immune response/rejection
- nucleases could potentially have off-target effects > cancer
- nucleases will continue to be made for years even when no longer needed
- expensive
- other ethical concerns: designer babies etc
23
Q
what factors to look for when choosing a vector
A
- origin of replication
- accommodate gene of interest and have multiple cloning sites (specific recognition sites) for endonucleases
- selective markers for screening - antibiotic resistance gene
- reporter genes (code for proteins which are easily measurable) - to assess success of DNA insertion (not essential tho)
24
Q
benefits of whole genome testing
A
- can see other underlying mutations and get much more info
- less time consuming and costly than lots of individual mutation testing
- increased understanding of how the whole genome works together
25
Q
cons of using animal insulin
A
- greater potential for rejection
- can have inconsistent composition and potency between batches
- inefficient procedure and low yield
26
Q
what does DNA fingerprinting (gel electrophoresis) rely on?
A
- use of gene sequences that are highly polymorphic (i.e. many variations of those genes exist in the population like SNP)
