Zinc Finger Nuclease – The new Technology for Targeted …€¦ · • Zinc Finger Nuclease...

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Zinc Finger Nuclease – The new Technology for Targeted Genome Modification in Animals

Dr. Rainer EbelSales Development Manager

Emerging TechnologiesSigma-Aldrich Corporation

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Agenda

• Zinc Finger Nuclease Technology

• Targeted Knockout Zebrafish

• Targeted Knockout/-in Rats and Mice

• Targeted Knockout in Rabbits

• Sigma-Aldrich

www.sageresearchmodels.com

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What is a ZFN?

A reagent that creates a very targeted double strand break (DSB) in genomic DNA


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ZFP Design via Single Zinc Finger“Mix and Match” Strategy

Fingers:

Triplets:

9bp target site:

3-finger ZFP:

+ +

Link fingers of known subsite preference to yield protein with desired composite specificity


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In most nucleases, DNA binding and nuclease functions are structurally integrated, making them difficult to engineer.

ZFN

FokI

Zinc Finger Nuclease


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Zinc Finger Nuclease Highly-Specific Genomic Scissors


Each homologous arm contains five to six Zinc Finger Proteins

Gene specificity is 30 or 36 bases

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ZFN-mediated Knockout


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Santiago et al, 2008, PNAS 105, 5809.

Genotype of Three New DHFR -/- CHO Cell Lines


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ZFN-mediated Knockin


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•Creating endogenous gene reporters

GFP-tubulin

GFP-actin

ZFN-mediated Knockin


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Agenda





• Sigma-Aldrich


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Animal Models: Somatic Disruption of Zebrafish ntl Gene


http://images.google.com/imgres?imgurl=http://www.biology-blog.com/images/blogs/12-2006/zebra-fish-61290.jpg&imgrefurl=http://www.biology-blog.com/blogs/permalinks/12-2006/how-does-a-zebrafish-grow-a-new-tail.html&usg=__IAXOqEheZkcm4sJN6E1xgsuEgL0=&h=302&w=411&sz=24&hl=en&start=4&sig2=n2Jkc8ThhkuhK5-XB-PbPA&tbnid=fV7BaLody83M5M:&tbnh=92&tbnw=125&prev=/images%3Fq%3Dzebrafish%26gbv%3D2%26hl%3Den&ei=O94NSrGoBoSkmQeAhriACA

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Animal Models: Somatic Disruption of Zebrafish ntl Gene

Injected ZFN mRNA into single cell embryoScored phenotype after 2 daysSequencing revealed broad range of deletions and insertions at targeted loci (single embryo alleles shown)>60% of founders carry ntl mutationsGermline frequency 20% (average)


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Agenda





• Sigma-Aldrich


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Historic Limitations in Genome Manipulation

Recombinant Knockout Technology

• Utilizes selectable markers to identify recombination events

• 12 – 18 month timeframe for founder creation

• Well established technology in mice for specific strains

http://nobelprize.org/nobel_prizes/medicine/laureates/2007/adv.html


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Knockout Rats Using ZFNs


http://www.mcw.edu/MCWHome.htm

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The World’s First Targeted Knockout Rats

Mutant x WT : Confirmed germline transmission (4/13 pups)

Pronuclear injection of ZFN against GFP into fertilized 1-cell stage embryos of GFP-rats

~ 12% efficiency


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p53 Knockout Rat

ZFN Assembly Time: 6 weeksZFN Injection: week 7Founders Born: weeks 10 – 11

– 15/58 Pups Born have Mutation– Identify by PCR and sequence analysis

From Gene Sequence to Founder Identified in 12 weeksFounder Sex Deletion Size Sequence

ttctccagtcttcctccagatgatattctggtaaggagccgg

53 male 5 ttctccagtcttcctccagat--------tctggtaaggagccgg36 female 9 ttctccagtcttcctcc---------------tctggtaaggagccgg

15 ttctccagtcttc----------------------tggtaaggagccggg37 female 3 ctccagtcttcctccagat--------attctggtaaggagccgg38 female 14 ttctccagtcttcctccaga-----------------------agagccgg40 female 11 ttctccagtcttcctccag------------------gtaaggagccgg33 male 3 ttctccagtcttcctccagat-------attctggtaaggagccgg18 male 9 ttctccagtcttcctcc---------------ttctggtaaggagccgg6 female 11 ttctccagtcttcct-------------------tccggtaaggagccgg

74 male 8 ttctccagtcttcctcct-------------tcctggtaaggagccgg


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p53 Validation by Western

(PAB240)

non-specific bandp53

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50

37

WT 731RP KO 733RP

5037

K L K LActin


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p53 Pathology

a. b. c.


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Zinc Finger Nuclease Knockout Mouse

•ZFN Assembly Time: 6 weeks•ZFN Injection: week of 4/21/09•Founders Born: 5/12, 5/13

– 34/44 Pups Have Mutation– Mutations Sequenced– Knockouts range from 3 to 695bps

9/9 Founders passed the knockout to F1s

Founder ID # Deletion size (bp)

2 53 44 35 6466 6957 198 24811 69113 39217 20

1918

18 219 2320 1921 53323 39625 53326 11

53427 75

1920

29 31231710


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Weinstein et al, 2010, Nat. Biotech


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2.01.5

1.0

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HA-L HA-R

PF PRNotI

1.81

0.840.97

1 2 3 4 5 6 7 8MKb

0.5

The Not I site was also integrated into the rat Mdr1a site using a similar donor construct

Targeted Integration of NotI into PXR in Rat Embryos


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Agenda





• Sigma-Aldrich


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PLoS One. 2011;6(6):e21045. Epub 2011 Jun 13.

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The Rabbit as a Model

• Cardiovascular disease• AMD• Dermal• Joint

*


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Microinjection Statistics

Session 1 Session 2 Session 3 Session 4 Total

Embryos Collected 207 119 120 111 557

Embryos Microinjected 196 98 96 75 465

Embryos Transferred 122 76 68 60 326

Rabbits Born 9 1 2 4 16

Embryos collected/rabbit born 35Embryos microinjected/rabbit born 29Embryos transfered/rabbit born 20

5/16 founders were identified (mutations ranging from 8bp to ~300bp deletion (some with insertions).


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• Sigma-Aldrich

Agenda


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• CompoZr® Targeted Integration Kit 2.125 €• Human AAVS1• Murin Rosa26

– Rapid Gene Insertion into the Human or Murine Cell Line of Your Choice– ZFNs engineered to target human AAVS1 or murine Rosa26 site

• Knockout ZFN (human, rat, mouse) 5.100 € (Plasmid only)– Production time: 3 – 12 weeks

• Custom ZFN against human, rat, mouse, hamster 12.000 € (Plasmid only)– Best performing custom validated ZFN against gene of interest– Production time: 9 – 12 weeks

• Custom ZFN against “non-standard species” 12.000 € (Plasmid only)– Up to Top three ZFN against gene of interest– Production time: 9 – 12 weeks (up to 6 months)


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Available from SAGE Labs

• Priority access to catalog knockout rat models

• Access to SAGEspeed™ custom model creation platform– Rat / Mouse / Rabbit– Founder animals (rat or mouse) produced in as little as 5 months– Limited researcher ‘slots’ available to customers per Year

• ModelShare™ Program– First of its kind rat model repository

• ADME/Tox• Mdr1a• PXR• Mrp1• Mrp2• Bcrp

Immunodeficiency• Rag1• Rag 2• DNAPK• Foxn

Carcinogenicity• p53

• CNS• ApoE• Leptin• BDNF• DISC1

• Pink1• Parkin• α-synuclein• DJ-1• Lrrk2


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Looking Beyond the Rodent…..


http://images.google.com/imgres?imgurl=http://mattcbr.files.wordpress.com/2009/01/goat.jpg&imgrefurl=http://mattcbr.wordpress.com/2009/01/24/man-tries-to-steal-car-turns-into-goat-gets-arrested/&usg=__2bu26YKwXqYJ79A7j2D2_84sEH0=&h=333&w=325&sz=128&hl=en&start=13&sig2=zENjAbPsvIGFVrbABkSODw&tbnid=PRVrlMQxiHwbzM:&tbnh=119&tbnw=116&prev=/images%3Fq%3Dgoat%26gbv%3D2%26hl%3Den&ei=CN4NSvaLDeaEmAf0it2ZCA

http://images.google.com/imgres?imgurl=http://www.historyforkids.org/learn/economy/pictures/chicken.jpg&imgrefurl=http://fatknowledge.blogspot.com/2007_04_01_archive.html&usg=__BqCiB5_GVgRh5iwwSoeOZ350Y-E=&h=386&w=360&sz=26&hl=en&start=4&sig2=CVwpYJvlOlyrP1L5lWlosQ&tbnid=PMZ7VM0B92l8-M:&tbnh=123&tbnw=115&prev=/images%3Fq%3Dchicken%26gbv%3D2%26hl%3Den&ei=6N0NSvfiI5OlmQfamLidCA

http://images.google.com/imgres?imgurl=http://ocw.usu.edu/University_Extension/sheep-and-lambing-management/sheep.jpg&imgrefurl=http://ocw.usu.edu/University_Extension/sheep-and-lambing-management&usg=__nHsujwx4BUVB5AMgRXY6fiaiJok=&h=500&w=500&sz=154&hl=en&start=1&sig2=tr4rbFPFVgQtB33swpIlcw&tbnid=SEaJIODMqdKJeM:&tbnh=130&tbnw=130&prev=/images%3Fq%3Dsheep%26gbv%3D2%26hl%3Den&ei=It4NSujTH-GGmQeUg8iZCA

http://images.google.com/imgres?imgurl=http://www.bbc.co.uk/jersey/content/images/2005/11/24/jersey_cow_350x350.jpg&imgrefurl=http://www.bbc.co.uk/jersey/content/ecards/jersey_cow_ecard.shtml&usg=__FzkpGePmfNpFjF3vrxv6lr3c8bs=&h=350&w=350&sz=21&hl=en&start=21&sig2=jNgKmdHbHs8vEVdR4UsNDw&tbnid=Y-tCaeT_sNx0oM:&tbnh=120&tbnw=120&prev=/images%3Fq%3Dcow%26gbv%3D2%26ndsp%3D20%26hl%3Den%26sa%3DN%26start%3D20&ei=uN0NSs6vGeWwmAew2dHTDA

http://images.google.com/imgres?imgurl=http://afeatheradrift.files.wordpress.com/2009/04/pig.jpg&imgrefurl=http://afeatheradrift.wordpress.com/2009/04/27/short-takes-on-the-day-042709/&usg=__R4jgI3iBrLl8q1eTTcD0nVTX9Ac=&h=345&w=387&sz=33&hl=en&start=77&sig2=4v0pc9idqaHPlUfGsK5dAA&tbnid=F4Bp_BfnkRj5gM:&tbnh=110&tbnw=123&prev=/images%3Fq%3Dpig%26gbv%3D2%26ndsp%3D20%26hl%3Den%26sa%3DN%26start%3D60&ei=gt4NSp_UJI-HmQfqusGFCA

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Dr. Rainer EbelSales Development Manager Central EuropeEmerging

Technologies

0172 / 83 64 [email protected]

Zinc Finger Nuclease – The new Technology for Targeted …€¦ · • Zinc Finger Nuclease...

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