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Evo Bio > EB20 > Flashcards

EB20 Flashcards

1
Q

How many deaths per year from Malaria

A

100s of thousands
mostly infants
mostly in Africa

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2
Q

what are current interventions for malaria and how effective are they
- whats the most optimistic scenario

A

nets spraying, drugs, saved millions but not enough to eliminate, resistance may lead to reversal and cost more than funding available.
most optimistic scenario (cost up to 9 billion a year) still leaves malaria in 62 countries by 2030

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3
Q

what are current innovations for malaria treatment

A

diagnostics, drugs, (one dose cures), vaccines, (75% efficiency allowing 80% coverage, implied 60% transmission reduction) and insecticides to maintain current levels in face of resistance will not be enough for eradication

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4
Q

what is the biological background of malaria

A
  1. Largely in rural africa
  2. 4 spp. of plasmodium causing: falciparum, vivax, malariae adn ovale.
  3. most transmission in Africa by A. gambiae/colluzii, arabiensis and funestus.
  4. species can also transmit O’nyong’nyong virus and filiariasis.
  5. vast maj of mosquitos dont transmit disease, only females bite and transmit
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5
Q

what could be two uses of gene drive in malaria

A

population suppression: reduce mosquito no.

population replacement: change mosquito so can no longer transmit

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6
Q

How are trasngenic mosqutios made

A
  1. plasmid and helper RNA micro injected into freshly laid eggs with a fine needle.
  2. many eggs die but some survive and incorperate DNA into their genome,
  3. injected adults are crossed to WT, larvae screen for RFP to identify transgenic
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7
Q

What is population suppression: Sex ratio bias of mosqutios theory

A
  1. based on naturally occuring driving Y chromosome in Aedes. - visible in crossing experiments
    —–when male is making sperm at meiosis breaks down X
  2. gradually convert population so it is predominantly male: dont bite so dont transmit.
  3. experiments make 95% males with normal fertility.
    only functional sperm will carry y chromosome = males progeny.
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8
Q

Describe the method of Y chromosome drive in Anopheles (IC research)

A
  1. enzyme made to cut X specific sequence (.e.g ribosomal repeat found only on X, in 100’s of copies).
  2. HEG coding Ppol enzyme inserted into Y chromsoome region - with control region flanking enzyme (beta 2 tubulin promoter), which is only expressed at meiosis so gene only expressed then.
  3. enzyme labelled wth GFP.
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9
Q

what did the trials of the IC anopheles Y chromosome drive show
how was it fixed

A
  1. first attempt didnt work as instead of making fertile males with a male sex bias, made sterile males.
  2. Enzyme made in testes was incorperated intt sperm and deliverred to zygote which cut up X chromsoome in zygote.
    - fixed by reducing the half life of the enzyme so wouldnt be active in zygote.
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10
Q

Describe gene KO by homing

A
  • can be used to gradually target gene in mosquito
    1. cell heterozygous for homing endonuclease gene makes an enzyme that cuts recognition site for HEG in chromosome, uses homologous chromsoome as template for repair and gene gets copied across, so heterozygote goes to homozygote.
    2. recognition site is only found in chromosome not containing the gene, the gene is in the middle of its own recognition site.
    3. nuclease gene can be spread through the population by same method as HEG and recognition site is a fertility gene.
    4. HEG will cause population wide KO of target gene.
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11
Q

where are HEGs found

A

found anturally in fungi, algae, protists and viruses but not animals.

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12
Q

describe the 3 basic steps in homing KO of fertility genes

A
  1. HEG males released and transmit HEG to most progeny
  2. mating of carriers prooducing homozygotres.
  3. homozygous male is fertile and continues to transmit HEG whereas homozygous female is sterile.
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13
Q

first demonstration of homing in animals

A
  1. taking homing endonuclease gene and recognition site from yeast.
  2. strong deviation from 50:50 inheritances, spread through a small cage population.
  3. high CRISPR based homing rates at female fertility genes. 94% across all genes.
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14
Q

What three endonucleases have been trialed to target mosquito genes

A
  1. TALENs
  2. HEG
  3. CRISPR/Cas
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15
Q

describe TALEN endonuclease

A
  • Fok1 cleavage domain can be linked to site specific recognition domain in TALENs: easy to engineer, binding/cleavage sites dont overlap, repetative structure.
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16
Q

describe HEG endonuclease

A

highly specific, found in primitive organiss, can be re-engineered to target DNA. efficient and highly specifci naturally select to home binding cleavage sites overlap, takes time to re-engineer.

17
Q

describe CRISPR/Cas endonuclease

A

can be guided to its target site by a short guide RNA

easy to make enzyme that can recognise and cut a sequence of DNA> binding cleavage sites overlap.

18
Q

what is the time course to elimination predicted

A

Expected 3-4 years after release

19
Q

Describe the TARGET MALARIA technology

A
  • product is a bucket of male mosquitos carrying a gene coding for a DNA nuclease
  • genes will spread through the target population over time and suppress/modify populations to reduce vectorial capacity and malaria transmission.
  • reducing burden of disease and facilitate other elimination programmes
20
Q

What are the intended key features of TARGET MALARIA technology

A
  1. reduce transmission.
  2. widely applicable: urban/rural, high/low disease, indoor/outdoor biting, irresepctive of available health system, stability of government, location of communities (access).
  3. area wide and regional control, long lasting compatible with and helpful to other interventions; no cross resistance.
  4. safe and inexpensive.
21
Q

What is a biological precedent for mosquito gene

A

control of Cassava mealybug.

  1. Cassava is a stable crop to SA grown widely in Africa
  2. bug came over from SA and caused devastation.
  3. Parasitoid wasp that only attacks mealy bug released.
  4. Large redution in bug population in short time.
  5. overall benefit cost ratio was good.
  6. self spreading nature as in gene drive, biological control
22
Q

Why may mosquito gene drive not work

A

males wont compete with WT males

  • replenish lab stocks with natural varaition to keep it close to whats our there. = soluble problem.
  • genetic construct itself is unlikley to cause problems as it is only switched on in testes.
23
Q

potential harms if gene drive does work

A
  1. possible extinction of mosquito species
  2. disruption of ecosystem
  3. empty niche filled by other vectors for disease
  4. plasmodoum evolves to other vectors
  5. mutations in nuclease gene after release.
  6. Resistance to mutation
24
Q

How do mosquitos mate in nature

A

males form swarms and and females fly into them, choose a mate, then fall out of swam. males are only alive for 4-5 nights.

25
Q

what is the harm in possible exctinction of mosquitos

A

moral

likely a population will be kept in the lab so wont ever be fully extinct, captive breeding programme.

26
Q

how could mosquito gene drive disrupt ecosystem

A

Pollinator: sweep net to see whether they have pollen, care mostly if its a specialist pollinator.
Prey: examine gut content or feaces of suspected predators, take samples and do DNA barcoding of region of mtDDNA and amplify by PCR. - see what fraction diet is due to mosquito species.
** can test manipulatively impose suppression and see what happens.

** nobody has currently reported ecological consequenes

27
Q

implications of empty niche being filled by something else

A
  • malaria is prevelent in Africa can be related to efficiency vectors: several years ago A. arabiensis accdientally intro to SA. - more efficient than local mosquito leading to outbreak.
  • potentialy african vectors better as evolved with humans longer.
  • can therefore nassume that if african vectors eliminated SA vectors would not fill their niche as not as good. eliminating the worst, means nothing worse can follow.
28
Q

what is the implication of plasmodium evolving to other species/organisms

A

lab selection experiment
seems unlikely people are bittne all the time by culex and aedes so any mutant that can transmit plasmodium would give plasmodium a selective advantage, but has not occured in nature.

29
Q

what are the implications for mutations in nuclease gene after release

A 
function KO is not a concern
other sorts of mutation: mutate insert into region dont want, e.g. insertion in immune system could make them better vectors.
30
Q

what are the implications for resistance to gene drive mutation

A
  1. mutations in target site no longer recognised by endonucklease
  2. for resistance: target essential sequence so cant change and still maintain function
  3. could have multiple guide RNAs targetting three different places
  4. could get selection for things not recognised by the enzymehttps://www.brainscape.com

Evo Bio (18 decks)

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