(Cyto)Selling non-human probes - OGT€¦ · found on the long (q) arm of human chromosome 7, major band 3, minor band 1 (Rommens et al. 1989). The third is from the perspective of

(Cyto)Selling non-human probes

Darren K Griffin

University of Kent

Chromosome Analysis Tools

Cytogenetics: Karyotyping

REO’Connor

2

karyotypesofseveralspecies(pig,human,birdandmouse)areillustrated.Ineachcase,an

imageofastainedmetaphasepreparationistaken,photographedandthechromosomes

arrangedaccordingtoaconventionagreedbythescientificcommunity.

Figure1-2:RepresentativeG-bandedKaryotypesfor(a)Pig,(b)Human,(c)AmericanRheaand(d)Mouse.

Therearethreeprimaryreasonsforstudyingakaryotype:thefirstisthatdeviationsfromthe

speciesnormareusuallyindicativeofdisease.Forthisreason,thehumankaryotypeismore

intensivelystudiedthananyotherspecies,withcataloguesofchromosomedisordersbeing

beyondthescopeofevenverylargevolumesofprint-basedmedia(Borgaonkar1975;Schinzel

2001).Thesecondisforthepurposesofgenomicmapping:ineveryeukaryoticspecies,each

geneinthegenomeislocatedwithreferencetothechromosomeonwhichitresides,thearm

ofthechromosomeandtheappropriatedistancefromthechromosomeend;forexample,the

cysticfibrosistransmembraneregulatorgeneisdefinedasbeing7q31,thismeansthatitis

foundonthelong(q)armofhumanchromosome7,majorband3,minorband1(Rommenset

al.1989).Thethirdisfromtheperspectiveofgenomeevolution(Ferguson-Smith&Trifonov

2007):giventhateachspecieshasauniquekaryotypeandthatmorecloselyrelatedspecies

usuallyhavesimilarkaryotypestooneanother,anychangesinthekaryotype,andtherefore

Molecular Cytogenetics:Fluorescence in situ hybridisation (FISH)

Genome Sequencing and Visualisation

(Evolution Highway – Murphy et al. 2005)

Microarray

What is FISH still used for?Humans

• Study and/or clinical diagnosis of

– Birth defects

– Cancer

– Infertility

– Gene mapping

– Sex determination in embryos

– Basic research

– Evolution (zoo FISH)

What is FISH still used for?Non-humans

• Comparative genomics– Zoo-FISH

• Genome assemblies• “Clinical” cytogenetics

– Cattle, pigs– Translocation screening– Cancer

• Cell lines • Tumours e.g. dog-cat

• Basic research– Chromosome behaviour– Nuclear organization– Cancer models

The big idea

• Develop a range of animal fluorescent probes• Mouse

– Basic research• “Mouse Octochrome”

• Pig/Cattle– Fertility and reproduction– Evolution

• Bird– Evolution – Agriculture – Basic research

• “Chicken multiprobe”

• £200,000 KTP grant – Two thirds of money came from BBSRC

The big idea







Mouse Karyotype

Trust me, this is hard

Mouse Chromosome Octochrome device

1. Spot slide with cell suspension

2. Multiprobe device spotted with hyb. buffer

3. Invert sample slide over multiprobe device

4. Overnight hyb

5. Rapid formamide-free wash

New product “Mouse Octochrome”

1,4,14 2,5,15 3,7,16 6,8,17

10,9,18 12,11,19 X,13,Y

Mouse Octochrome

1,4,14 2,5,15 3,7,16 6,8,17

10,9,18 12,11,19 X,13,Y

The big idea







Screening for Hypoprolificacy in Pigs using Subtelomeric FISH Probes

Rebecca O’Connor

1

6

2 Current Screening

4

3

4 The Multiprobe™ Kit

1 Background

3 Subtelomeric Probes

5 However…

6 Conclusions

1 Background

6

2

5

4

3

• Reciprocal translocations

– reduced litter size/hypoprolificacy

• Increasing emphasis on AI

– significant cost of using hypoprolific boars

• Current screening

– karyotyping

– simple but requires animal specific expertise

• FISH based assay reduces need for this level of expertise

Background

• Hypoprolific boars:– phenotypically normal – normal semen profile

• Translocations:– seen in around 0.47% of boars awaiting service (Ducos et al, 2007)– can be de novo in origin

• Boar to sow ratio: ~ 1:20 in natural mating ~ 1:200 in AI

• Litter sizes up to 25-50% smaller

Background

Huge potential economic cost of not screening!

1

6

2 Current Screening

5

4

3

• Identification of aneuploidy and translocations

• Cryptic translocations difficult to identify

• Requires animal specific expertise

• We actually do several hundred of these a year

• 30 Translocations found in 500 boars screened

Porcine karyotype derived from cultured lymphocytes

Current Screening

• Existing karyotyping service for ACMC and Danish Agricultural and Food Council

• 500 samples karyotyped to date and 30 pigs with translocations found

Current Screening

1

6

2

5

4

3 Subtelomeric Probes

FISH: Whole Chromosome PaintingSubtelomeric Probes

- BACS isolated from the subtelomeric region of the p-arm and q-arm for each autosome and the X chromosome

- Directly labelled with FITC or Texas Red

(Knight and Flint, 2000)



Reciprocal Translocation 38, XY rcp(4;9)(q-;q+)

1

6

2

5

4 The Multiprobe™ Kit

3

• Incorporation of the subtelomeric probes into the Multiprobe™ device (Cytocell Ltd.) to enable all chromosomes to be analysed on one slide

• Each square carries subtelomere specific probes for both the p-arm and the q-arm of each chromosome

• Improves speed and accuracy and therefore reduces cost

Telomere LocalisationThe Multiprobe™ Kit

1pq 2pq 3pq 4pq 5pq 6pq 7pq 8pq


17pq 18pq Xpq

Pig Subtelomeres device


9pq

10pq

11pq

12pq

13pq

14pq 15pq 16pq

17pq 18pq Xpq


Practical application of the Multiprobe™ Kit:




4. Overnight hyb



• 1 boar from JSR Genetics showing fertility problems

• No discernable chromosome abnormality by regular Karyotyping

• Multiprobe device detected a 5:6 translocation

•

•

From JSR: Translocation Screening

• Boar Identified

– 61 Litters produced• Litter size 4.8 pigs less

– £10,248

• Lost premium on 183 gilts (culled not sold)

– £10,980

– Financial Cost £21,228

– + reputational cost

– + logistical cost (unable to fulfil orders)

1

6

2

4

3

5 However…

The Role of H2BFWT

Progress was delayed as a significant proportion of the Pig BACs did not map correctly…..

– Total BACs tested: 79

– Of which: 35 initially mapped correctly and 44 map incorrectly (44% correct)

– 91% of those mapped incorrectly (i.e. not on end of chromosome),

• Most of these were on the right chromosome, but in the wrong place

However…..

The Role of H2BFWT

Should map to distal end of 15q

However…..

The Role of H2BFWTHowever…..

• Results shared with Alan Archibald (from the Swine Genome Sequencing Consortium)

– errors appear to be due to assignment of the smaller fingerprint contigs “randomly shoved on the end”

• All probes now correctly mapping and multiprobe device has gone into production

1

2

5

4

3

6 Conclusions

Conclusions

• The Multiprobe™ Device and FISH probes:- enable simultaneous detection of whole pig karyotype on one slide

• Porcine translocation screening is:- significantly faster

- more cost effective

- more accurate

- no need for animal specific expertise

- And can detect cryptic translocations

• Results from this study will be incorporated into the next iteration of the pig genome assembly

Commercial Product Development Future

• Cattle device

• Human Probes that will work on all mammals

Translocation Screening - Cattle

• Cattle 2n = 60 – all acrocentric and similar size

– Really difficult to karyotype!

Translocation Screening - Cattle

• Subtelomeric translocation screening probe is developed

Bos taurus Chr 24 (p-arm Red; q-arm Green)

REO’Connor

112

Figure3-8:SubtelomericcattleprobesforBTA24p-armlabelledinFITC(CH240-382F1)andq-armlabelledinTexas

Red(CH240-19L13).Scalebar10μm.

Afurtherchickenspecificdevicewasdevelopedwhichisdiscussedinthenextchapter.

3.5 Discussion

3.5.1 ChromosometranslocationdetectionusingFISH

Resultsfromthisstudyprovideproofofprincipleofanapproachthatcanbeusedsuccessfully

todiagnosechromosomaltranslocationsthatdirectlyimpactfertilityinpigsataresolution

previouslydifficulttoachievebystandardkaryotyping.Itwasalsoextendedtocattleand

chicken.Therearethreeadvantagesofusingthisapproachoverclassicalkaryotyping:Thefirst

isthatitdetectsmorecryptictranslocationsthanstandardkaryotypingotherwisewould.The

boarwitha5:6reciprocaltranslocationdescribedinsection3.4.4isanexample.Indeed,thefact

thatapreviouslyundetectedcryptictranslocationwasidentifiedwouldsuggestthattheactual

numberoftranslocationsintheboarbreedingpopulationmayinfactbesignificantlyhigherthan

previously reported. It is possible that these karyotypically cryptic and unreported

translocationsareseenmorefrequentlythanexpectedbutthattheroutineuseofmultiple

inseminationspersowmaybedilutingtheeffectonthefarrowingrates.Theboarwithacryptic

The big idea







Homo sapiens Gallus gallus

• Majority of chromosomes are tiny microchromosomes

• Microchromosomes occur in reptiles (lizards, snakes, turtles)

• “So many, so small” pattern is uniquely avian feature

The Avian Karyotype

• This is what you find in most publications

From Schmid et al., Cytogenet Genome Res (2005)

This is a lot harder than a mouse

The Avian Karyotype

Practical application of the Multiprobe™ Kit:

Chicken Multiprobe device




4. Overnight hyb



• Chicken Multiprobe device

– Macro chromosome 1,2,7,Z in FITC

– Macro chromosome 4,5,6, W in Texas red

– Macro chromosome 3,8,9 in Aqua

– Micro chromosome 10-28 p and q arms in FITC and Texas Red


1,4,3 18pq 2,5,8 19pq 7,6,9 20pq Z,W 21pq

22pq 23pq 24pq 25pq 26pq 27pq 28pq


GGA 16 GGA 17 GGA 18

GGA 19 GGA 20 GGA 21Gothami Fonseka, Becky O’Connor, Pam Lithgow, Deborah Smith


Comparative genomics

Chicken

Budgerigar

Gyr falcon

FISH – Avian Cytogenetics

• Set of ~250 FISH probes developed to work across 21 avian species

• FISH protocol optimised to improve cross species hybridisation

• Edited ‘cross-species panel’ of high success FISH probes developed – 2/3 per chromosome

Chicken Chromosome 1

Hybridisation Success Rates

230 FISH Probes – ‘Selected’ and ‘Non-selected’ tested on 5 diverse avian species to evaluate phylogenetic tree coverage

Hybridisation Success: Peregrine Pigeon Zebra Finch Chicken Turkey

Chicken selected 93% 92% 91% 100% 100%

Chicken non-selected 47% 26% 21% 100% 89%


• Cross-species panel tested on 10 species so far with close to 100% success

• Results show an exceptional degree of (previously undetectable) genome conservation


Next Steps:

• Cross-species panel to be tested on reptiles

Turtle and Lizard FISH!

‘Protomicrochromosome’ - ancestral macrochromosome region that split in avian lineage to form a microchromosome (chicken chromosome 26)

Lizard Turtle

Became a University of SharjahPractical Class

Sharjah practical class

The big idea







Sales to date

• In 2014– 20 Viral DNA integrated chicken micro and macro chromosomal devices (Prof.

Venugopal Nair, Pirbright Institute)– Zebra finch BACs (Griffin Labs, £1200 towards KTP consumables expenses )

• In 2015– 35 Chicken Multiprobe devices (Central Veterinary Research Laboratory, Dubai )– 60 Mouse Octochrome devices (Dr. Aurora Ruiz Herera, Universitat Autònoma de

Barcelona, Spain)– 3 Mouse Octochrome devices+ FISH (Dr. Karen Keeshan, University of Glasgow)– 100 Pig subtelomere devices (Griffin Labs/JSR genetics collaboration project) – 4 Mouse Octochrome devices (Dr. Peter Ellis, University of Kent)– 10 Chicken Multiprobe devices (University of Kent)– 5 Chicken Multiprobe devices (Christ church University) – Mouse liquid paints (from various customers, orders through production) – 15 Chicken devices – 30 pig paint tests 7FITC/10 TxR (University of Kent)– Mouse Chromosome Paints ( Dr. Peter Ellis)– 6 mouse Octochromes (Dr. Peter Ellis)– 282 in total

In 2016-17

• Our KTP project received an “outstanding” report• Pigs

– JSR Genetics • 100 pig devices per year

– TOPIGS NORSVIN• Norway, Holland and Spain

– Up to 300 per year (eventually)

– Current 150 per year for both the above

• Cattle– Genus PIC

• Initially was potential for 500-2000 cattle devices per year– Depending on if we see some reciprocal translocations– Sadly they have put this one on ice

• Bird– Dubai students

• 15-20 bird devices

– Griffin lab and collaborators

Benefits to Cytocell – new products

• Pre-existing products– Pig device

• Up to 300 per year

– Chicken• 30 ish per year

– Mouse• Continuation

• Products in advanced stages of development– “Any bird” device– Cattle

• The company we were chasing a little less interested than they were

• Future – “Any mammal” human probes– MyProbes service for people in non-human cytogenetics

Benefits to Cytocell - other

• Presence at conferences– ICACG, ICC, ECA

• Press

• Staff training – Possible PhD projects

• Pairs of hands for– “Semi-risky” projects

• Joint grants – LINK grant not funded first time but has been resubmitted

– Can we discuss another KTP?

Projects currently in train

• Improved FISH technique– Selection criteria (Joana, Becky)– Optimised protocols

• Greater proportion of probes working more often

– Multiprobe devices (Becky, Gothami, Anjali)– Faster FISH buffers (Jacob, Becky)

• Very expensive faster buffer works well• Trying to re-create it

• Semi-automated capture (Henry, Jacob, Ben)• Generally working in principle• Works well for good signals• Still needs some tweaking for “weak” signals• Liaison with Digital Scientific UK

– SmartCapture 4 now in production – Dovetail with Multiprobe/Octochrome device

New grant application• Select panel of ~600 human BACs from Cytocell collection

– To work on any mammal metaphase

• Generate draft chromosome-level genome assemblies for 5-6 pilot mammalian species,

• Design a Multiprobe device and pipeline to achieve multiple hybridisations– ~70 BAC clones per slide)

– Drafts of chromosome assemblies for 40-45 mammals

• Adapt protocols/tools for the screening of pigs and cattle

• Upload to browser

• To address several biological questions and hypotheses

• Publicly facing web site

Dr Gothami Fonseka

• Did a PhD involving contract work for the Bridge Centre and Digital Scientific to generate results for her thesis and supplement her income

• Awarded her PhD in 2013

• Raised £8,000 towards her final year fees from the Kent Cancer Trust and £5,000 for consumable money for her project

• Has presented at charity events and international conferences

• Has published 6 papers

• Was the researcher on the KTP project with Cytocell

• Now part of the Cambridge team

C SR

CENTRE FOR INTERDISCIPLINARY STUDIES

OF REPRODUCTION

What is CISoR?

• A lot of academics and their commercial partners from different disciplines with a lot in common

• A working on something to do with the following1) Assisting human reproduction2) Barriers to human reproduction3) Non-human reproduction

• In the following areas1) Inter-disciplinary research 2) Impact, knowledge exchange and enterprise 3) Communication, policy, public debate, consultation, media; 4) Education, training and skills

• Finding an excuse to get together– Often, and an enjoyable way– Networking

• Collaborative ventures that might not otherwise have happened– Such as academic-industrial projects

• Celebrate achievements• The whole will be greater than the sum of the parts

Documents

(Cyto)Selling non-human probes - OGT€¦ · found on the long (q) arm of human chromosome 7, major band 3, minor band 1 (Rommens et al. 1989). The third is from the perspective of