W5: Transposon tools for insertional mutagenesis and targeted expression

Transposable elements

• ‘transposons’

• account for most dispersed repetitive DNA in eukaryotic genomes

BUT ALSO

• really useful in the lab!

Why useful in the lab?

1. Mutagenesis

2. Insertional mutagenesis

3. Ease of crossing

4. Enhancer trapping

1. Mutagenesis→ why useful

• large scale unbiased mutant screens→ great for dissecting mechanisms of cell function and development

  1. Generate loads of random mutations in genome

  2. screen for occasional mutations that affect process of interest

     • e.g cell division, embyronic development, cell death, behaviour etc

  3. any mutant that affects this must have hit important component of process

1. Advantages of this approach

• no need for preconceptions about

  • nature of protein, cellular location, enzymatic properties

• Only need informative phenotype

1. Disadvantages of this approach

• Following up interesting mutations needs→ cloning of gene

  • route to molecular analysis of its function

e.g If used Chemical metagenesis

• generates many point mutations

  • and affected gene must be identified by ‘positional cloning’

    • easier than it used to be but not trivial

2. Insertional mutagenesis (compared to chemical mutagenesis→ why better approach!)

• mutation caused by insertion of DNA fragment into gene

• this fragment carried a molecular ‘tag’

  • can be easily identified by mutant phenotype

  • much easier to clone and analyse molecularly

    • → than a gene in which only point mutations are available

3. Ease of crossing of these mutants

1. if transposon carriers a dominant marker

   • (e.g white+→ expressed from its own promoter)

   • → it is easy to follow the insertion in genetic crosses

2. If carried a recessive marker

   • e.g mutant phenotype caused by the insertion

   • → not as easy!

4. Enhancer trapping

Consider:

• transposon construct with reported gene fused to minimal promoter

  • e.g→ GAL4(DBD): encodes DNA binding domain (DBD) of the yeast transcription factors GAL4

• significant transcription only occurs when the element integrates next to the enhancer in the genome (figure)

• As most enhancers are cell-type specific

  • → different insertion sites of an ‘enhancer -trap’ element therefore allow many cell types to be labeled by GAL4 expression

4. It may also be possible to identify…

• a nearby gene that shows the same expression pattern as the GAL4 reporter

why:

• because it is driven by the same enhancers

In order for transposons to be useful in the lab

• need transient controlled transposition

• so mobile DNA can insert at new genomic locations at a high enough frequency

Drosophila P transposon

1. encodes Transposase

   • → needed for tranposition of the element

2. Transposase binds to specific sequences near end of P element

3. together with endogenous Drosophila proteins→ catalyses

   1. excision of element

      • and

   2. sometimes transposition

Therefore: P transposon will always EXCISE element→ but not always put it in somewhere else!!

Partially deleted P element without transposase can still transpose if

1. There is another source of transposase

2. it has cis-acting sequences

   • that are necessary to bind transposase

3. has other necessary host proteins

A single intact transposase gene anywhere in the genome can…

• Catalyse transposition→ in trans

  • of any other P element which has all the needed cis-acting sequences

A partially deleted P element which lacks intact transposase gene (but with all needed cis-acting sequences for transposition) will…

• remain stably integrated→ in the absence of transposase

  • will NOT tranpose further

Using these principles in the practical

• Used to recover new insertion of a P Element

• Element used:

  • enhancer tap construct

    • THEREFORE

    • will also assess whether any new insertions recovered are giving novel patterns of GAL4 gene expression

    • Thus→ potentially detecting novel cell types and genes and generating genetic tools to manipulate these cells

  • Plus acquiring and optimising fluorescence microscopy images

Actual Experiment stuff:

How to identify new P element insertions

Experimental background→ cross of flies set up

Females→ wild types

Males→ carry

1. P construct P{w+, DBD-ET}

   • carries white+ gene inserted on a 2nd chromosome (CyO→ Curly of Oster)

   • which is marked by Curly (Cy)

2. Mutation in endogenous w gene on X

   • THEREFORE: only w+ is on the P element

     • (so can follow the P element)

3. CyO→ ‘balancer’ chromosome

   • carries multiple inversions which when heterozygous with wild-types chromosome→ prevent recovery of recombinants in the progeny

     • HOWEVER: (not needed just yet) coz male Drosophila=no crossing over

       • but still needed for females in other parts of crossing scheme

4. Third chromosome→ with tightly linked (so make sure know where transposase is)

   • P transposase gene

   • and dominant marker→ Dr (Drop)

What these modifications to the male fly allow

• Mobilisation of P elements in the male parents

  • If happens in germline cells→ progeny could contain new P insertion (or just excision) in all of their cells

  • If transpoase expressed in somatic cells→ still some insertion/excision BUT not transmitted to progeny

    • (so not interested)

THEREFORE: by studying the progeny of this cross→ can see what these new insertions/excisions look like!

What are the next steps in this practical

1. Identify individual flies that carry new P insertions→ new transposants

2. Cross these to partners (with flouresencne gene)

3. Obtain larva to examin for expression of GAL4(DBD) enhancer trap

Deciding how to identify new transposants: Questions to ask

• Q1a. Work out the genotypes of the gametes produced by each parent in the above cross

• Q1b. Work out all the classes of progeny - their full genotypes and their phenotypes (including their sex) - of the above cross. A Punnett Square may help you to do this. For now, don't (yet) worry how transposition may affect this.

• Q2. Your next task is to work out how to spot occasional flies that MUST carry a new insertion. To work towards this:

  • Q2a. Should you keep or discard flies with the original P insertion? How can you do this?

  • Q2b. Should you keep or discard flies expressing transposase? Why? How can you do this?

  • Q2c What flies will carry neither the original P insert, a new P insert, nor transposase?

  • Q2d. Finally, which flies (the ones that you want) carry neither the original insert nor transposase, but do carry a new insert?

  • P.S. Does it matter whether you select males or females?

Q1a: work out genotypes of gametes produced by each parent in the cross

Just mix all the ways to combine together with punnet square

Q1b: Work out all the classes of progeny

Progeny phenotypes

1. Curly, red, drop

2. Curly, red, WT

3. WT, white, Drop

4. WT, white, WT

Half male and half female

Q2a: Should you keep or discard flies that carry the original P insertion? How to do this?

If progeny:

1. Curly→ will carry P{w+} too

   • means has original insertion and NOT a new one (not of interest!)

   • → IGNORE: curly red-eyed

What to look for: flies with non-curly wings

Q2b. Should you keep or discard flies expressing transposase?

• DO NOT WANT

• Why:

  • we want to keep the new insertions made stably integrated and non-mobile

  • if tranposase is still expressed→ new insertions might be lost/move again

• HOW TO GET RID

  • Keep only Dr+ flies

    • Coz Dr marker is on transposase-producing chromosome

Q2c. What flies will carry neither the original P insert, a new P insert, nor transposase?

• white-eye, non-curly which are not Dr

THEREFORE: get rid

Q2d. Which flies carry neither original P insert nor transposase, but carry a new P insert?

• Red eyes, non curly, not Dr

What is the genotype of the vigin females

w; +; UAS-GFP, elav-AD

• homozygous for UAS-GFP and elav-AD constructs

  • → (homologous chromsome pairs are separated by semicolons)

• This will provide the florescence for visualistion of this progeny

Q3: Could different transposant individuals carry identical or non-identical insertions? For this answer, does it matter whether they came from different vials or the same vial? Hint: does the stage of development when transposition occurs matter?

1. DIFFERENT VIALS: New transposants from different vials are very unlikely to carry insertions at the same location

   • why: P element can integrate throughout the genome

2. SAME PARENTAL VIAL:

   • may sometimes also applies

   • BUT OFTEN: carry the same new insertion

     • WHEN: if transposition occurred early enough in the germine for mitotic cell division to have produced many germ cells (with the same insertions)

THEREFORE: depends on timing of the insertion event if all have same insertion in the same vial

4. Could mobilisation occur in somatic cells of the male parents? Does this matter?

• transposase is expected to express in somatic cells of the male parent

but

• this will have no influence on classes of progeny obtained

  • coz somatic cells do not contribute to gametes

5. When the progeny of your crosses hatch as adults, what proportion of the progeny do you expect to carry an insertion?

• Progeny carry a 1:1 ratio of new insertion to no insertion

• coz ust a 50:50 chance

  • The most common event is single insertion of P{w+, DBD-ET}

    • which is heterozygous

    • THERFORE: 50:50 chance

6. Why will some insertion events be mutagenic and others not? Why will insertion into a gene normally cause a recessive mutant phenotype?

1. some insertion events will be in genes (or their regulatory sequences)

   • → THEREFORE: mutant genotypes

2. others will be in intergenic regions which will

   1. not inactivation of any gene→ NO phenotype

   2. some will inactivate genes not necessary for viability→ cause phenotype that is not immediately obsvious to a superfial inspection

      • → YES PHENOTYPE BUT NOT SEEN (that is really seen)

Insertion of P element will probably inactivate the gene

• supplying a copy of the wild-type gene will usually supply enough gene product to produce WT phenotype

  • → THEREFORE just recessive

7. Sometimes mobilisation excises the P insertion. Some excisions appear “precise”, some leave some P element sequences present, and some excise flanking genomic DNA.

• How could you identify any progeny that carry such excisions?

• Why might excision of flanking genomic DNA sometimes be useful for us?

• White-eyed, Curly flies

  • lost the w+ but still have curly CHECK THIS

• Excisions of flanking DNA may sometimes generate useful delections of nearby genes

8. How could you establish a stable stock of any new insertions? Think: How can you maintain a stock of an insertion that is homozygous lethal?

Principle→ from existing knowledge:

• simplest stable sock is one homozygous for the insertion

How to get this?

• crossing two heterozygous parents for the same insertion

• BUT: initial transposant male fly (red-eyed non-curly non-Drop male_ cannot e crossed to itself

• THEREFORE: must first cross it to generate F1 flies

  • HALF: will be heterozygous

• THEN cross these heterozygous males and females to eachother

Must need a way to identify heterozygous and homozygotes

Way to identify heterozygous and homozygotes

Need suitable genetic markers

• Provided by balancers→ e.g CyO for insertion on a second chromosome

For this example of 2nd-chromosome insertion:

1. cross heterozygous w+ insertion fly x w- balancer stock (w;CyO/If)

   • If→ dominant eye shape marker

THEREFORE

2. in F1→ half will be heterozygous for the w+ insertion and half will carry no insertion

   • of w+ F1 flies→ half will also carry CyO and be {w+}/CyO

3. Cross these heterozygous males and virgin females to each other

4. 25% of their progeny will be homozygous

   • → can be identified as non-curly

How can you maintain a stock of an insertion that is homozygous lethal?

• If it is maintained heterozygous with different homozygous lethal mutation

  • → only heterozygotes are viable

But this only works indefinitely if you…

• suppress the recombination between the two homologous chromosomes

  • → need to prevent recombination giving rise to homozygous viable recombinants that carry neither the lethal insertion nor the homozygous lethal mutation

How can this be done?

• having your insertion heterozygous with a balancer chromosome

  • → which also carries a homozygous lethal mutation (or mutliple)

Procedure of W5 practical

Given 3-4 vials of progeny from fist cross, need to

1. Check you are finding all classes of progeny that you expect to find

2. Screen for any flies with new insertions

3. set up crosses as described, we provided partners

Recognising phenotypes and markers

1. Eye colour markers

   • w+- red, w- white

   • w+ inserts→ may only be pale red, yellow, orange

     • not always expressed at high enough level for WT

2. 2nd Chromosome markers

   • Curly→ dominant wing phenotype

   • BEWARE: can be mild and hard to distinguish from WT

3. 3rd Chromosome markers

   • Dr (Drop)→ greatly reduced eye

   • easily scored BUT don’t mistake lack of normal-sized red eye for w-

4. Sexing flies

   • Male→

     • black patch on the dorsal posterior abdominal segments

     • one segment fewer than females

     • sex combs on the front legs of the males (reliable indicator)

USE microscopes for this to make sure!

Things to be cautious of

1. do not over-anaesthetise→ use minimum CO2

2. do not allow to stick onto the food

3. Do not bang tube on bench→ might bang food out

   • hold together and tap hands gently onto the bench

4. Dispose of unwanted flies into morgue asap→ avoid cross-contamination

5. Each cross wit single new insertion (enough just one insertion→ see above) and note if from same or different vial

6. Use virgin females→ because females will store the sperm