Genetic transformation of insects

Question 1

Why is genetic transformation needed?

Answer 1

Would be useful as part of integrated vector management as resistance to current insecticides is rapidly increasing.

Question 2

What is one way to genetically develop sterile insects? What was a problem with this?

Answer 2

Use chromosome inversions and select a trait of insecticide resistance where treatment with the insecticide would kill the females but leave the males. Effective but toxic to the environment. PRoblems were also with not being able to rear enough numbers.

Question 3

What is the P element?

Answer 3

Transposible elements in drosophila that cause hybrid dysgenesis and were seen to spread round the world very quickly.

Question 4

Descrive the process of microinjection. How effective is it?

Answer 4

• Insert gene of interest into a transposible element.
• Use a needle to inject into embryo.
• Protien binds ot inverted repeats and inserts the plasmid into the genome.
• Can use a fluorescent marker to visualise difference beween WT.
• Less effective than CRISPR, only 1% may be infected.

Question 5

What are the two ways in which genetic transformation can be applied for control purposes?

Answer 5

-Population suppression
uses a lethality gene to cause a population crash
-Population modification
changes disease transmission dynamics

Question 6

Describe Oxitec’s first and second-generation population reduction strategies.

Answer 6

First-Kills both males and females

Second-only kills females

Question 7

What technology does population replacement rely on?

Answer 7

Gene drive to allow greater rates of inheritance rapidly compared to mendelian genetics.

Question 8

What are the 4 main techniques that can be used to increase copy number of genes?

Answer 8

• Homing endonuclease genes
• Transposable elements
• CRISPR
• Zinc finger

Question 9

Describe homing endonuclease as a genetic transformation tool

Answer 9

Gene produces an endonuclease which cuts DNA on the opposite chromosome. This is repaired naturally and therefore produces two copies (it is now homozygous). Produces homozygosity without the need for sexual reproduction/ recombination.

Increases the copy number.

Question 10

Describe daisy drive as a genetic transformation tool

Answer 10

Linear chain of sequentially dependent unlinked drive elements. C drives B drives A etc (can be on separate chromosomes). ELements at the base of the chain (i.e C), are lost over time to naturally lost over time due to natural selection as they do not have a driver themselves.

Spreads the gene but also controls the spread.

Question 11

Describe transposable elements as a genetic transformation tool. How does piggyback work?

Answer 11

Non specifically inserts into the genome (e.g piggyback inserts at TTAA which is almost anywhere in the genome).

Can inject into female eggs or ovaries to increase copy number.

Question 12

What is medea? What does it stand for and how does it work?

Answer 12

Maternal effect dominant embryo arrest.
Is a selfish gene for a maternal toxin and a zygotic antidote. The mother expresses the toxin in the germline to kill the progeny. If the offspring also carry medea, they produce the antidote and survive. Because it is a selfish gene, it gives it a selective advantage over normal genes.

If introduced into populations at high levels, it will replace entire populations to those carrying medea.

Can link medea to a gene of interest e.g. one for malaria resistance as a way of controlling insect-borne diseases.

Question 13

How does CRISPR cas9 work for genetic transformation?

Answer 13

Based on a bacterial defence system. Allows the cell’s genome to be cut and that sequence replaced or that sequence removed in vivo.
Cutting induces homology-directed repair of the strand opposite to where the (e.g.) malaria resistance gene

Question 14

What are some challenges with some gene-editing technologies such as zinc finger, crispr, transposable elements etc?

Answer 14

• Hard to control once released
• Are not self-limiting and transposable elements can cross between species (rare but not impossible).
• Unknown stability in the field (e.g. interaction with other transposable elements and genomes)
• Hard to get regulatory permission for testing
• Variable environmental consequences with mosquitos in urban populations

Question 15

What is the oxitec population reduction approach?

Answer 15

Release genetically modified males which mate with wild females and their offspring die before reaching adulthood.
Uses a marker (actin 5 c promoter) and a a self limiting gene (HSP70 minimal promoter which is preceded by TETO)

Without tetracycline, the gene enhances its own transcription which produces the TTAV protein which is toxic at high concentrations (by inhibiting transcription factors for multiple genes, therefore, causing cell death). With tetracycline, the TTAV protein is blocked from binding to TETO which switches off its positive feedback and reduces the production of the TTAV protein.

Question 16

Which mosquitos does adulticiding target?

Answer 16

Male mosquitos

Question 17

To compete with the mating of WT males, how many GM males need to be released?

Answer 17

10 to 100 times as many GM males to WT males. This is due to mating efficiency disparity.

Question 18

What are the three phases of Oxitec’s approach for testing and implementing GM to crash mosquito populations?

Answer 18

Phase I: test the system by finding unique sites on the X chromosome. Insert an endonuclease into the unique site. It cuts the X chromosome leaving it non-functional. It destroys the X chromosome in the sperm and the X in the egg upon fertilisation. Modified males produce no progeny.

Phase II: Make it self limiting (modification only passed on for a few generations) to test how it spreads and manipulate sex ratios. Shreds the X in sperm but modified so it doesn’t shred the X in the egg on fertilisation. Produces predominantly male offspring. Self-limiting because females have more breeding opportunities than males so males are at a competitive disadvantage and the trait is selected out over time.

Phase III: Self-sustaining. put endonuclease onto the Y chromosome so that modified males only pass on a functional Y chromosome so 100% of offspring are male = population crash

OR can reduce female fertility by introducing a nuclease which disrupts a female fertility gene, causing sterility if both copies of the gene are disrupted.