(cyto)selling non-human probes - ogt€¦ · found on the long (q) arm of human chromosome 7, major...
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(Cyto)Selling non-human probes
Darren K Griffin
University of Kent
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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
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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)
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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
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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
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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
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Mouse Karyotype
Trust me, this is hard
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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
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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
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Mouse Octochrome
1,4,14 2,5,15 3,7,16 6,8,17
10,9,18 12,11,19 X,13,Y
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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
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Screening for Hypoprolificacy in Pigs using Subtelomeric FISH Probes
Rebecca O’Connor
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1
6
2 Current Screening
4
3
4 The Multiprobe™ Kit
1 Background
3 Subtelomeric Probes
5 However…
6 Conclusions
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1 Background
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2
5
4
3
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• 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
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• 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!
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1
6
2 Current Screening
5
4
3
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• 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
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• Existing karyotyping service for ACMC and Danish Agricultural and Food Council
• 500 samples karyotyped to date and 30 pigs with translocations found
Current Screening
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2
5
4
3 Subtelomeric Probes
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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)
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FISH: Whole Chromosome PaintingSubtelomeric Probes
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FISH: Whole Chromosome PaintingSubtelomeric Probes
Reciprocal Translocation 38, XY rcp(4;9)(q-;q+)
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2
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4 The Multiprobe™ Kit
3
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• 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
9pq 10pq 11pq 12pq 13pq 14pq 15pq 16pq
17pq 18pq Xpq
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Pig Subtelomeres device
1pq 2pq 3pq 4pq 5pq 6pq 7pq 8pq
9pq
10pq
11pq
12pq
13pq
14pq 15pq 16pq
17pq 18pq Xpq
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Telomere LocalisationThe Multiprobe™ Kit
Practical application of the Multiprobe™ Kit:
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
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Telomere LocalisationThe Multiprobe™ Kit
• 1 boar from JSR Genetics showing fertility problems
• No discernable chromosome abnormality by regular Karyotyping
• Multiprobe device detected a 5:6 translocation
•
•
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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)
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2
4
3
5 However…
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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…..
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The Role of H2BFWT
Should map to distal end of 15q
However…..
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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
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6 Conclusions
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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
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Commercial Product Development Future
• Cattle device
• Human Probes that will work on all mammals
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Translocation Screening - Cattle
• Cattle 2n = 60 – all acrocentric and similar size
– Really difficult to karyotype!
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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
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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
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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
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• 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
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Practical application of the Multiprobe™ Kit:
Chicken Multiprobe 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
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Chicken Multiprobe device
• 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
10pq 11pq 12pq 13pq 14pq 15pq 16pq 17pq
1,4,3 18pq 2,5,8 19pq 7,6,9 20pq Z,W 21pq
22pq 23pq 24pq 25pq 26pq 27pq 28pq
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Chicken Multiprobe device
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GGA 16 GGA 17 GGA 18
GGA 19 GGA 20 GGA 21Gothami Fonseka, Becky O’Connor, Pam Lithgow, Deborah Smith
Chicken Multiprobe device
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Comparative genomics
Chicken
Budgerigar
Gyr falcon
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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
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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%
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FISH – Avian Cytogenetics
• Cross-species panel tested on 10 species so far with close to 100% success
• Results show an exceptional degree of (previously undetectable) genome conservation
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FISH – Avian Cytogenetics
Next Steps:
• Cross-species panel to be tested on reptiles
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Turtle and Lizard FISH!
‘Protomicrochromosome’ - ancestral macrochromosome region that split in avian lineage to form a microchromosome (chicken chromosome 26)
Lizard Turtle
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Became a University of SharjahPractical Class
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Sharjah practical class
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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
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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
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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
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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
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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?
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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
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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
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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
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C SR
CENTRE FOR INTERDISCIPLINARY STUDIES
OF REPRODUCTION
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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