Lecture 10 - Genetic Screens in Higher Eukaryotes

Question 1

What are examples of multicellular model organisms?

Answer 1

• C. elegans
• Arabidopsis

Question 2

What are C. elegans?

Answer 2

• a small transparent nematode round worm
• 1mm long, transparent, contain 900 cells
• in the wild it lives in soil and eats bacteria
• can be lab-grown on plates or in liquid culture (& fed E. coli)
• grows quickly (egg-to-egg in 3.5 days). Lives for 3 weeks
• Can reproduce sexually (1000 progeny) or by self-fertilizing (300-350)
• genome is 100Mb, 5 autosome pairs & sex chromosome
• efficient transgenics
• genomic resources - WormBase

Question 3

What is the life cycle of C. elegans?

Answer 3

• From Egg to Larvae 1 (L1) stage: it takes 8 hours from the laying of egg to L1 stage. The whole embryogenesis from sperm entry to hatching takes about 14 hours.
• The Dauer stage: C. elegans develops into dauer larvae stage under starvation conditions.
• From Dauer to L4: after placing in food, dauer larvae will reach L4 stage.
• Its life cycle is about 3 days

Question 4

What occurs in the L2d stage?

Answer 4

L2d stage is when there are not sufficient nutrients around. It will travel to the L2 stage instead if there is sufficient nutrients.

Question 5

Why are some C. elegans hermaphrodites?

Answer 5

have sperm & egg

Question 6

What are the difference between Males & hermaphrodites?

Answer 6

Hermaphrodites:
- sex chromosomes are XX
- produce both eggs & sperm
- mostly self-fertilize
- produce 99% XX (1% XO)

Males:
- sex chromosomes are XO
- rare in the wild but can be bred in the lab
- produce only sperm
- mate with a hermaphrodite to produce 50% XX & 50% XO.

Question 7

How do the 1% XO in hermaphrodites occur?

Answer 7

Through non-disjunction in humans

Question 8

What are hermaphrodites not?

Answer 8

Female worms

Question 9

What are Worms a model for?

Answer 9

• Eukaryotic development
• Post-genomic sequence (genome was sequenced & shared in 1998)
• Apoptosis - shown that this can be a deliberate act
• Cell signalling
• Aging
• RNA interference (RNAi)

Question 10

What is Wormbase?

Answer 10

A site facilitating insights into nematode biology

Question 11

Describe how to create a homozygotes mutants using classic genetic screens in C. elegans

Answer 11

• Parental strain (both alleles wild-type - for gene of interest) - P0
• Worm treated with mutagen (e.g. EMS) which might create mutations in egg, sperm or body
• Hermaphrodite self-fertilize and creates F1 generation - heterozygous for mutant
• As it is diploid, phenotype not always seen
• F1 self-fertilize & creates 1:2:1 (wild-type: heterozygous: mutant)
• 2 copies of mutant gene = mutant type
• select mutant & put on fresh plate to self
• As it is homozygous for mutant allele - all progeny will show mutant phenotype

Question 12

How do you make transgenic worms?

Answer 12

• DNA can be directly injected (along with a selectable marker) into the gonad.
• The DNA can be linear or circular
• Can knockout a gene or add a transgene using homologous recombination
• In worms, the selectable markers are phenotypic - not antibiotic. They have to be dominant (needs to be dominant as there is only 1 copy)
• One common marker is a dominant collagen mutant called rol-6
• A roller worm looks like this

Question 13

What is the characterisation of RNAi in worms?

Answer 13

RNA is a way of reducing gene expression by triggering the degradation of the mRNA

Injecting works with double-stranded RNA (dsRNA) complementary to exon sequence sequences (important as it is targeting mRNA - introns already removed) results in specific silencing of the gene.

The silencing spreads through the organism

The silencing is inherited into progeny (whilst they are still inside the mother)

Silencing doesn’t occur in animals defective for RNAi

This makes it much easier than making transgenic mice

Question 14

What is RNAi (RNA interference)?

Answer 14

A process by which double-stranded RNA silences specifically the expression of homologous genes through the degradation of their cognate mRNA. In worms, a gene can be selectively disabled and its phenotype determined simply by feeding wild-type animals double-stranded RNA.

Question 15

How does RNAi work well in worms?

Answer 15

• RNAi is an endogenous cellular process by which messenger RNAs are targeted for degradation by double-stranded (ds) RNA of identical sequence, leading to gene silencing
• RNAi can be used to make knock-down mutants in worms
• for specific gene-knockdowns it is more efficient to use the injection method
• For large-scale screens, long dsRNAs are fed to the worms in E. coli
• These RNAi screens were used to characterize cell-to-cell signalling during development

E. coli with plasmid with 2 T7 promoters (phage (virus) injects bacteria). 2 promoters - 1 goes clockwise & 1 going anti-clockwise.

A multiple cloning site in the middle. Whatever gene in the multiple cloning site - creating 2 copies. This creates double-stranded DNA E.coli then fed to worms

Question 16

When do you use the injection vs feeding method?

Answer 16

If you know what gene - injection method

If you don’t know what gene - feeding method

Question 17

What are genome-wide RNAi screens?

Answer 17

• you can purchase RNAi feeding libraries
• 16757 clones

Question 18

What is the difference between RNAi vs classical mutant screen?

Answer 18

Classic genetic screen:
- gain-of-function alleles can be isolated, which can uncover regulatory mechanisms
- tissue-specific alleles can be recovered
- insights into structure - function relationships can be obtained from point mutations
- every gene should be mutable using this approach
- cloning stage in laborious
- maternal-effect genes with zygotic requirement are hard to identify
- mutations usually affect single genes
- mutant alleles are heritable

RNAi screen:
- RNAi-mediated knockdown results in a reduced levels of wild-type product
- not every gene is susceptible to RNAi - some tissues are resistant and genes encoding proteins with long half-lives are hard to knock down effectively
- the gene sequence is known immediately
- Can introduce double-stranded RNA at different developmental, by bypassing earlier requirements
- multiple genes with shared sequence can be knocked down, thereby uncovering redundancy
- knockdown is usually not heritable, except when the silencing construct is expressed as a transgene(s)

Question 19

What is Arabidopsis?

Answer 19

• Arabidopsis thaliana is a small weed belonging to the brassica family (related to cabbages).
• It is easy to grow (for a plant) and has a rapid life cycle (6 weeks), abundant progeny
• this genome is 135 Mb (over 5 chromosomes - no sex chromosomes - encoding about 27,000 genes)
• Each gene is about 2kb with 4 introns

Question 20

What is Arabidopsis a model for?

Answer 20

• Evolution & adaption (varieties can be found globally - meaning phenotype can be linked to specific environment)
• Population genetics
• Development of more complex plants such as maize & wheat
• Environmental interactions (light, water, disease)
• Plant genomics
• Gene regulations

Question 21

Why is Arabidopsis a good model?

Answer 21

• Short life cycle (6 weeks seed-to-seed)
• Large number of progeny (see rare events)
• Can be grown in restrictive conditions
• Can be efficiently transformed using Agrobacterium tumefaciens
• Genome sequence available (since 2000)
• Large collection of mutant stocks, natural ecotypes & genomic resources

Question 22

Describe the life cycle (genetically) of Arabidopsis

Answer 22

• Plants do not have a germ line
• Both diploid & haploid cells undergo mitosis
• In higher plants (including Arabidopsis), the haploid phase (or gametophyte) is reduced to just a few divisions in the ovule (egg) or pollen.
• In ancient plants like ferns or mosses, their haploid phase is bigger & sometimes dominant.

Question 23

Describe Arabidopsis mostly self-fertilizes, but is amenable to crossing

Answer 23

Take male & female plant. Demasculate flower (remove stamen) - preventing self-fertilization. Tie cotton around plant to remember who male is. Take tweezers and take part of plant & touch the top - transferring pollen. Leave to develop. Paper bag over to collect seeds.

Because it self-fertilizes, it is useful as homozygous lines can be stably maintained.

Crosses can be performed when required (e.g. mapping genes, backcrossing to parental lines, looking at the interaction between genes).

Question 24

How does evolutionary diversification & adaption occur?

Answer 24

• population genetics
• can study natural variation
• 1001 Genomes Project was launched in 2008 to discover detailed whole-genome sequence variation in at least 1001 strains (accessions).

Question 25

Describe transgenics in Arabidopsis

Answer 25

Inside agrobacterium there is a plasmid (T-plasmid) - T for transfer. Small portion of T-plasmid is injected from the bacteria into plant cell & integrated into genome. You cannot choose where the DNA goes - RANDOM INTEGRATION. It might land inside central gene & kill the plant.

Question 26

How do you generate a T-DNA insertion population?

Answer 26

Take T-DNA (with selectable marker - e.g. herbicide resistance gene). Put gene of interest in plasmid & put the plasmid inside agrobacterium. Plant then dipped in solution with agrobacterium - T0. The seeds collected & grown on selectable marker. Each one of the resistance seeds in an independent T1 line - T-DNA has integrated inside a different spot inside the genome. Each seeding will have an independent T-DNA insertion. Some of these will be in genes. This is likely to abolish gene function in the gene that it is inserted into (make a null mutant) Can insert tagged genes, such as GFP. Insertion into random locations

Question 27

How do we know where the T-DNA is?

Answer 27

Large collections of T-DNA insertion mutants are available.

Question 28

What are Arabidopsis resources?

Answer 28

- Mutant collections - Stock centres - Genome information - Epigenome information - Expression profiling

Question 29

What are big discoveries in Arabidopsis?

Answer 29

- Arabidopsis is definitely considered THE model plant in the post-genomic era. - It is commonly used as a model for crop plants - It is handy for looking at development of multicellular organisms because (unlike animals) the cells never migrate or move.

Question 30

What are 3 things that Arabidopsis is famous for?

Answer 30

1. Flower development, homeotic mutants & the ABC model 2. Vernalization & epigenetic silencing 3. RNA silencing

Question 31

What is an example of Functional Analysis by gene discovery?

Answer 31

1. Amass mutants affecting the biological property of interest 2. Cross the mutants to wild type to see if their descendants show ratios of wild to mutant that are characteristic of single gene inheritance 3. Deduce the functions of the gene 4. Deduce how the gene interacts with other genes to produce the property in question

Question 32

What are the steps in screening Arabidopsis?

Answer 32

1. Perform a forward genetic mutant screen 2. Cross back to the parental strain 3. Identify the gene responsible by mapping 4. Verify that we have the correct gene by ordering a null mutant from a stock centre & looking for the phenotype we observed in our screen. 5. Find out more about the new gene, and plan our next experiment using information from publicly available databases.