Invertebrate Biotechnology Flashcards
What is a transgenic insect?
Insects are made transgenic when one of more DNA sequences from other organisms are inserted into their genome
What is the accepted method of making transgenic insects?
Splicing the DNA intended for insertion into a mobile element also known as a jumping gene
What is a jumping gene?
A segment of DNA that can be integrated at many different sites along a chromosome
Describe the first stable transformation of an insect
1982
Drosophila
P transposable element used as the vector for transformation
Why are Drosophila used as the model system?
Have well characterised genetics
Have a short life cycle (10 days at room temp)
Easy to culture to large numbers
Easy to screen for effects of chemical mutagens
What are the disadvantages of using Drosophila?
Have poorly understood digestive biochemistry
What are mobile genetic elements?
A type of genetic material that can move around within a genome, can be transferred from one species, rearrange genes in the host genome, can cause mutations in protein coding regions (alter protein function) or replicon to another and play a distinct role in evolution
Are found in all organisms and in humans ~50% of the genome is thought to be MGEs
What are some examples of mobile genetic elements?
Transposons
Retrotransposons
DNA transposons
Insertion sequences
Plasmids
Bacteriophage elements
Group I and II introns
Who discovered transposons?
Barbara McClintock who found that maize plants had higher than expected mutation rates due to their different colours
Transposons were detected by their genetic effects as re-insertions can cause mutations
Mazie has 10 types of transposons
What is a transposon?
A short (3-4 kb) DNA sequence that can change position within a genome, sometimes creating or reversing mutations and as such can alter a cell’s genome size
Are classified into different families according to their sequence similarities
Contribute to a large part of plant genomes
What are the 2 classes of transposons?
Class I (retrotransposons/copy and paste)
Class II (cut and paste)
Describe class I transposons
Require RT
Can be autonomous (contain the sequence to encode its own transposase thus moves itself)
Can be non-autonomous (lack the gene for transposase or RT so must borrow these proteins from another element in order to relocate thus requires an active element to move and as such are more stable)
Describe class II transposons
Encode a transposase for excision and insertion
Can be autonomous and non-autonomous
Describe autonomous transposons
Autonomous elements contain a gene for transposase endonuclease that allows excision and re-insertion
Describe non-autonomous transposons
Are in 2 parts in the host genome
The transposase cuts the mobile element out upstream
The mobile element contains terminal repeats
What are the 3 schools of thought surrounding the origins of transposons?
Originated in the last universal common ancestor
Arisen independently multiple times
Arisen once and then spread to other kingdoms by horizontal gene transfer
What is the link between viruses and transposons?
Are found in all major branches of life
May confer some benefits on hosts but are generally considered selfish DNA parasites
Like viruses they live within the genome of cellular organisms, share features in their genome structure and their biochemical abilities
Speculation transposons and viruses may share a common ancestor
What is the link between bacteria and transposons?
Excessive transposon activity can destroy a genome so many organisms including bacteria have developed mechanisms to reduce this activity
i.e. high rates of gene deletion to remove transposons and viruses from their genomes
Transposons also may act as mutators in bacteria
How do eukaryotes manage transposon activity?
May have developed RNAi
In C. elegans nematode some genes required for RNAi reduce transposon activity
What is the link between transposons and vertebrates?
They may have been co-opted by the vertebrate immune system as a means of producing antibody diversity
What are P elements?
A family of transposons in Drosophila
First appeared in the species in the mid 20th century and have since spread through every population
Describe the first use of P elements to create Drosophila mutants
1982 Rubin and Spralding
Artificial P elements used to insert genes into Drosophila by injecting the embryo thus exogeneous DNA sequences introduced into the Drosophila germ line
A rosy transposon (ry1) was constructed by inserting a chromosomal fragment containing the wild-type rosy gene (red eye colour) into a P element
This transformed between 20-50% of the injected embryos
The transformants contained 1 or 2 copies of chromosomally integrated intact ry1 that were stably inherited in subsequent generations
Transformed flies had the visible genetic defect in the mutant host strain and were permanently corrected by the transferred gene
Why aren’t P elements used outside of Drosophila?
Attempts to genetically transform non-drosopholid insects with P elements were not successful as it is not a universal transposon (Handler et al, 1993)
What is piggyBac?
A TTAA specific class II transposon which was originally discovered in the cabbage looper moth by Fraser et al in 1983
Is common among other animals than moths
Was identified in Baculovirus mutants which are a family of large rod shaped insect pathogens (immature larval forms of the moth species are the common host)
Was subsequently found to have originated in the genome of the insect species (Cary et al 1989)
How is Baculovirus acquired by insects?
Insect feeds on contaminated foliage
Viral particles are released from their occlusion bodies and attach to the brush border of the gut cells
The virus then replicated in the insect cells
What are TTAA specific elements?
A group of transposons that share a similarity in structure and properties of movement
Originally defined in Lepidoptera but are common among other animals
How does piggyBac move?
Encodes a function that facilitates its own movement which is supplied on a helper plasmid
Can transpose into the baculovirus genome in insect cells whilst carrying a marker gene (lacZ)
Movement can be demonstrated in cells from a lepidopteran species distinctly related to the species from which it originated
Can be used as a helper-dependent vector for the transfer of genes in insect cells and that its movement is not restricted to species of origin (Handler 2002)
What is the structure of piggyBac?
2.4 kb element
Has inverted terminal repeat sequences on the 5’ (TTAA) and 3’ (TTAA) ends which are important for intermolecular recombination and circularisation of adeno-associated virus genomes
Has 13 bp inverted repeat sequences
Has a 1.8 kb ORF which encodes a functional transposase
How does the piggyBac transposon system work?
piggyBac transposes between vectors and chromosomes via a cut and paste mechanism
During transposition, piggyBac transposase recognises transposon-specific ITRs located on both sides of the transposon vector
This efficiently moves the contents from their original sites and integrates them into TTAA chromosomal sites
This enables genes of interest between the 2 ITRs in piggyBac to be easily mobilised into target genomes
Describe the first use of piggyBac in Drosophila
Handler 1998
Used to change eye colour
What were the problems with using piggyBac?
An obstacle to the use of transposon mediated germ line transformation was difficulty in obtaining marker genes that allow for identification
How are marker genes produced for piggyBac vectors?
Species-specific transformation markers can be generated by isolating visible mutations in the species of interest, cloning the corresponding gene and rescuing the mutant phenotype by the incorporation of a wild copy of this gene by transformation however this process is very laborious for many species and delays the development of new procedures
What is an example of a universal marker gene that can be used with piggyBac?
GFP can be used to show gene transfer in any species under the control of artificial promoters
What are 4 physical methods for DNA delivery in insects?
Microinjection (most popular for egg development)
Biolistics
Electroporation
Lipofection
Describe how mobile genetic elements are used to transform the germ line of an insect
An appropriate vector system is chosen (i.e. class I/class II)
The vector is injected into developing insect embryos by microinjection
Why is it important to be able to genetically engineer other insects than Drosophila?
Allows engineering of pest species and vectors of disease
Engineering of pest species has been an important lab tool in analysing gene function and effects on phenotype
Describe and explain some livestock insect borne diseases
Flies such as screw worm flies, bot flies and warble flies cause damage by feeding on flesh
Flies such as house flies, blow flies and horse flies mechanically transmit diseases
Mosquitoes, midges, sand flies, blackflies and tsetse vector disease after a blood meal and can also carry bacteria, viruses, protozoa and filarial worms
These cause diseases such as African horse sickness, blue tongue, Rift valley fever and encephalitis
Describe and explain some human insect borne diseases
Mosquitoes, midges, sand flies, blackflies and tsetse vector disease after a blood meal and can also carry bacteria, viruses, protozoa and filarial worms
These cause diseases such as malaria, yellow fever, dengue fever, sleeping sickness and river blindness
Give some statistics about malaria
Between 300-500 million people contract it every year
~3 million die every year (most children under 5)
Kills more people than any other communicable disease except tuberculosis
Endemic in over 100 countries
Transmitted by female Anopheles mosquitoes after biting to obtain a blood meal
There is no effective vaccine as the Plasmodium parasite has multiple immunologically distinct developmental stages and effective immune avoidance strategies
What happens when a mosquito has been engineered to be more refractory?
Are less able to transmit pathogens i.e. malaria
Describe how the laboratory strain of mosquito Anopheles gambiae allowed for disease control through engineering refractoriness using immune genes
A. gambiae was found to have an increased ability to encapsulate Plasmodium as they emerged from the epithelial midgut into the haemocoel
Thus attention was focused on the innate immune system of the mosquitoes as a source of genes that could be used to introduce artificial refractoriness into transgenic mosquitoes however suitable promoters (allow expression of the immune response gene at the time the pathogen was present) needed to be identified
Vitellogenin promoter was expressed in response to a blood meal (Kokozoa et al 2000) thus controlled spatial and temporal expression
What is the plasmodium life cycle?
Gametocytes to gametes that mate to zygotes to ookinetes
Ookinetes cross the midgut epithelium and differentiate into sporozoites which are released into the haemocoel
Development of the parasite is complete when it crosses the salivary gland epithelium
Why is attacking Plasmodium favoured in the midgut?
Numbers of the parasite are at their lowest thus most vulnerable
What are the 2 genes used in engineering refractoriness?
SM1 which is midgut and salivary gland specific and driven by a carboxypeptidase promoter activated by blood which blocks the epithelia, reduces oocyst infection rates by up to 60% in Anopheles stephensi (Ito et al, 2001)
+PLA (bee venom phospholipase) which reduces oocyst infection rates by 87% and a midgut specific carboxypeptidase promoter was used to drive the expression in A. stephensi (Moreira et al, 2000)
Neither are 100% effective at blocking transmission
Describe how early sporogonic and developing oocysts of Plasmodium in A. gambiae can be made more refractory
The Streptomyces phiC31 site specific transgene system was used to express a synthetic anti-malaria gene Vida3 in the midgut which was expressed in the midgut of bloodfed females which reduced parasite intensity by 85% (Meredith et al, 2011)
What are some problems with engineering refractoriness?
Knowledge of whether Plasmodium is virulent to its vector and by how much is relevant to the success of genetic engineering
The fitness cost to mosquitoes of refractoriness imposed by transgenesis could be partly eroded by the benefit of hosts not becoming infected
What is SIT?
Sterile insect technique
Describe how Oxitec genetically modified Aedes aegypti 1st gen and why
Population suppression by releasing 3.3 million sterile male insects carrying a dominant lethal to control dengue
OX513A mosquitoes carried the LA513 transposon via piggyBac which encoded the tTA protein whose high-level expression is deleterious to cellular development thus is self-limiting and rendered mating events unsuccessful with wild-type females (95% progeny died before adulthood)
2009-2018 Cayman Islands trial unsuccessful
Describe how Oxitec genetically modified Aedes aegypti 2nd gen and why
OX5034
After mating, female progeny die whilst male survive and half carry the self-limiting gene whilst the other half carry natural insecticide susceptibility genes
May 2020 US EPA allowed Oxitec to produce 2.4 billion for release in Florida in 2024
Describe how transgenic mosquitoes were produced using CRISPR
Mutated the doublesex gene which is involved in sexual development in A. gambiae (Hammond et al, 2021)
Produced genetically female insects whose mouth pieces resembled male insects thus could not bite
Reproductive organs were deformed so couldn’t lay eggs
Briefly describe the history of silk
Shanxi between 4000-3000 BC where a silk cocoon was cut with a knife
Fragments of a loom found dated to 4000 BC
What is the link between moths and silk
Silk moth Bombyx mori is thought to have originated by domestication from B. mandarin 5000 years ago
Is of high economic value
Why produce silk using biotech methods?
Produce silk moths that are disease resistant
Improve silk production and quality
Increase the number of silk genes
Increase the larval developmental period to generate larger moths
Introduce spider genes to make special silk
Describe how larval growth in moths was extended
Zhang et al 2017
Juvenile hormone controls metamorphosis in insects and levels of this hormone are regulated by juvenile hormone esterase
JHE depletion extended larval periods in B. mori by activating the TOR pathway thus making giant pupae and cocoons
Done using CRISPR
What is special about spider silk?
Many species of spider have different glands to produce silk with different properties for different purposes i.e. housing, defence etc.
E.g. Argiope argentata has 5 different types of silk including dragline silk
What is dragline silk and why is it special?
Used in the outer rims of webs and is as strong per unit as steel but much tougher
Spidofibroin forms the building blocks of dragline silk
Mass production has not been feasible due to the fact that spiders can’t be domesticated due to their territorial and predatory nature
Describe how chimeric silk protein was made
Fused Spidofibroin and amino acid sequences involved in the transport and processing of expressing silk protein in moths
piggyBac was used to transform B. mori
Fibres spun by these transgenic moths are a hybrid of spidofibroin and Fib-H (moth silk)
To increase the proportion of spidofibroin in the silk, the amount of endogenous Fib-H was reduced using RNAi
Mass produced spider silk
Teule et al 2012
Why is the US military interested in spider silk?
Monster silk, a hybrid of spider and moth silk is flameproof, stronger than steel and absorbs > 100,000 joules of kinetic energy from blasts
It is essentially pure spider silk produced by monster silk moths that have been engineered to have red eyes for distinction with normal moths
