Post on 14-Jul-2020
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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
www.sageresearchmodels.com
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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
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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
• 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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Animal Models: Somatic Disruption of Zebrafish ntl Gene
www.sageresearchmodels.com
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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)
www.sageresearchmodels.com
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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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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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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
www.sageresearchmodels.com
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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
75
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
• 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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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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• Zinc Finger Nuclease Technology
• Targeted Knockout Zebrafish
• Targeted Knockout/-in Rats and Mice
• Targeted Knockout in Rabbits
• Sigma-Aldrich
Agenda
www.sageresearchmodels.com
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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)
www.sageresearchmodels.com
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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
www.sageresearchmodels.com
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Looking Beyond the Rodent…..
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Dr. Rainer EbelSales Development Manager Central EuropeEmerging
Technologies
0172 / 83 64 714rainer.ebel@sial.com