SIT & gene drive

Question 1

genetic control of vectors

Answer 1

• various definitions
• does not necessarily involve modified or transgenic organisms
• ability to introduce trasngene does improve prospects of genetic contorl of vectors of disease

Question 2

population suppression

Answer 2

• elimination or reduction of a wild vector population
• e.g. introduce large numbers of sterile males
• introduce selfish genetic drive element to destroy gene essential to the vector itself

Question 3

population replacement

Answer 3

• replace pathogen-susceptible vector population with pathogen-resistant insects
• e.g. genetic drive system spreads a desirable trait (e.g. in the form of a transgene) through a population

Question 4

SIT

Answer 4

• sterile insect technique
• mass-rear sterile males and release into the nevironment
  
  • matings unsuccessful → decrease in population size
• requires good knowledge of the insect
  
  • remating propensity
  • seasonality

Question 5

steps involved in SIT

Answer 5

• mass rearing insects (millions a week)
• sexing insects (physical/genetic)
• induce sterility (radiation/chemical/genetic)
• release insects
• measure dispersal and mating competitiveness (mark/recapture, sperm transfer)

Question 6

insect sexing

Answer 6

• physical separation
  
  • based on difference in pupal size
  • inefficient, labour intensive
• Y-linked translocation of insecticide resistance allele or temperature sensitive lethal mutation
  
  • breakdown fo linage under mass rearing conditions
  • reduced fertility
  • females die in presence of insecticide/raised temperature

Question 7

sterilisation by radiation

Answer 7

• gamma radiation - usually cobalt 60
• substantial genome damage, also in soma
• reduced fitness and mating success in males

Question 8

SIT in agriculture

Answer 8

• widely used
• fruit flies, onion fly
• cost $25 million in mexico but saves $3 billion
• new world screwworm
  
  • parasitic fly eats living tissue of warm blooded animals
  • eradicated in north america 1982-1996
  • sterile males released along panama canal to prevent re-immigration of mated south american females

Question 9

SIT in disease vectors

Answer 9

• tsetse fly in zanzibar
• mass release 1995
  
  • average 7000 males per week
  • last wild fly 1996
  • last case of nagana in cattle 1997

Question 10

advantages of transgenic methods over SIT

Answer 10

• improved sexing
  
  • modify males → GFP in testes
  • automated sorting machinery
  • divert males and females to separate compartments
• e.g. RIDL
  
  • offspring produced to create sustained effect on population

Question 11

RIDL

Answer 11

• release of insects with dominant lethal alleles
• aedes aegypti, oxitec (first), tTA/tetO system
• transgenic construct with (now) female specific promoter
  
  • drives tetracycline transactivator
  • binds operator sequence to switch on effector gene
  • effector gene is lethal → females die
• presence of antidote (tetracycline) inhibits transactivator
  
  • survival
  • susbequent removal leads to death
• homozygous males released → mate with WT females → female offspring have allele → female death

Question 12

limitations of SIT

Answer 12

• scale and cost of rearing
• insects need to be transported to release site
• fitness of SIT insects
• immigration from non-treated areas
• inundative technology
  
  • number of insects released proprotional to outcome
  • requires release in a lot of areas

Question 13

SIT & malaria

Answer 13

• little success with anopheles
• males released usually less competitive
  
  • disperse and die
• one example has succeeded in elimination
  
  • very small trial area

Question 14

non-inundative methods

Answer 14

• preferred
  
  • exponential effect on population
• cassava mealybug success in africa
  
  • parasitic wasp from south america
  • lays eggs in mealybug
  • 150 point releases of wasp
  • 10x reduction of mealybug within 4 years
  • no resurgence
  • wasp replication allows large effect

Question 15

genetically engineered mosquitoes

Answer 15

• technology has existed for 10 years to prevent malaria transmission
  
  • SM1 peptide developed, selectively binds midgut epithelia receptors
  • receptor used by parasite
  • SM1 competes with parasite for receptors
• SM1 transgene with 4 SM1 peptide units
  
  • promoter activated upon mosquito feeding
  • 80% inhibiton of oocyst formation
• challenge is population spread

Question 16

spreading transgenes

Answer 16

• main challenge of genetic control by engineering
• evidence suggests that cost of plasmodium infection is the same as the cost of expressing refractory gene
  
  • no advantage so no propagation
• genetic drive of selfish genes could be a solution

Question 17

selfish genes

Answer 17

• aim to cheat the process of meiosis so that the frequency of a transgenic allele increases
  
  • biased, non-mendelian inheritance
  • spreads through population even though no increase in fitness
    
    • = selfish genes
• aim of gene drive
  
  • TEs, MEDEA, HEGs

Question 18

TEs

Answer 18

• transposable elements (selfish genes)
• gene flanked by inverted repeats
• transposase binds own IRs to copy itself and insert randomly into another part of the genome
• to progress to next generation, needs to insert in germline genes (if in an animal)
• somatic cells → no change → no advantage to TE
• active during gamete formation to increase chance of inheritance in germ line

Question 19

TEs and population replacement

Answer 19

• insert anti-malarial gene e.g. SM! into TE construct
  
  • specific promoter
  • TE jumps around and carries genetic load with it
• in theory this works
  
  • P-element spread through drosophila by TE invasion

Question 20

drawbacks of TEs

Answer 20

• form a large part of mosquito genome
  
  • defence mechanisms common
• loss of genetic load
  
  • SM1 mutations can occur so the TE spreads on its own
  • probably spreads better
• highly active transgenic TEs have been hard to produce
  
  • made but only jump once in a few generations
• other techniques will work better

Question 21

MEDEA

Answer 21

• Maternal Effect Dominant Embryonic Arrest
• selfish MEDEA element
  
  • maternal toxin and zygotic antidote
• female produces eggs and deposits toxin in all eggs
  
  • deposited irrespective of inheritance
• if zygote hasn’t inherited MEDEA, no antidote produced → death
• heterozygous father → 25% of offspring die
• can engineer SM1 into element

Question 22

MEDEA frequency

Answer 22

• always selection against WT individuals without element
  
  • can’t survive toxin deposition in eggs
• MEDEA frequency increases with each successive generation
• initial threshold number of MEDEA individuals needed

Question 23

difficulties with MEDEA

Answer 23

• finding/deciding on toxin and antidote
  
  • in drosophila, toxin = microRNA targeting zygotic gene
  • antidote = copy of same gene without miRNA binding site → no inactivation of gene
• loss of genetic load of anti-malarial effector SM1
  
  • no reason to keep it - spread better without
• so far only succeeded in drosophila, not mosquitoes

Question 24

HEGs

Answer 24

• homing endonuclease genes
• highly specific DNA endonucleases
  
  • cut DNA at unique target sites
  • recognise sequence occuring once in a genome
  • something like 25bp long
• selfish genes

Question 25

HEG mechanism

Answer 25

• HEG on opposite chromosome to recognition site
• cuts recognition site
• ds break repaired using other chromosome as a template
  
  • this chromosome has HEG instead of the recognition site
  • HEG is copied over
  • 2 HEG copies instead of 1
• essentially parasites of DNA repair machinery

Question 26

HEGs engineered into mosquitoes

Answer 26

• take HEG from organisms like slime mould
• alter to recognise mosquito target site only present once in msoquito genome
• establish gene drive
• if homing occurs in germline cells before gamete formation
  
  • inherited by all progeny (copied to both chromosomes)
  • cheats meiosis (selfish gene)
• even heterozygotic offspring will become homozygotic after homing
• attach e.g. SM1 to spread it in population → replacement

Question 27

advantage of HEGs

Answer 27

• only found in unicellular organisms
  
  • no defence mechanism

Question 28

HEGs for population suppression

Answer 28

• place HEG inside essential mosquito gene
  
  • disrupt and inactivate gene
• HEG recognises sequence in essential gene
• specific promoter to activate in germ line
• homing and spread in population
  
  • mate with WT → heterozygous offspring
  • still 1 functional gene copy
• spread continues and frequency increases
• eventually carriers mate wiht each other → homozygous offspring → no functional essential gene so death
• population fitness decreases, size decreases populaiton eliminated
• self-propagating genetic disease

Question 29

sex ratio distortion

Answer 29

• startegy for population suppression
• mutate Y to always succeed in fertilisation
  
  • only males produced
  • female population crashes
  • eliminates population
• present in aedes and culex
  
  • poorly understood mechanism
  • breakage of X chromosome when chromosomes segregate in testes

Question 30

building a sex ratio distorter

Answer 30

• sequence for ribosomal genes only on X
• express in males endonuclease to cut X chromosome during meiosis
• only produced Y bearing sperm
  
  • 95% male offspring
• no females produced int he first place so no female killing
• currently only expressed in autosome
  
  • aim to make it express from Y (germ line)
  • self-sustaining to produce only males