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• Cancer (CAG-Floxed-Stop, KRASG12D, p53R167H; caCSN2-miBRCA1-RASSF1)
• Cardiovascular Disease (hFAT-1+; TEK-NOS3+; TEK-CA...
• Model of Infection for PRRSv
• SIGLEC1
• CD163
• Timelines & From Here to Where?
– Initial binding with heparan sulfate
– Binding/Internalization by Sialoadhesin
– Internalization/uncoating of the virus ...
Heparan Sulphate
Binding
Binding and
Internalization
by
Sialoadhesin
(SIGLEC1)
Uncoating of
the virus by
CD163
Genome Rele...
Southern Probe
Prather et al ‘13 J Virology
• A targeted clone (4-18) was identified and used both for
somatic cell nuclear transfer and to propagate for Southern
blo...
Enucleation
Fusion or
Injection and
Activation
Development
Transfect or
Transduce and
Select
Synchronize?
Nuclear
Transfer...
SIGLEC1+/- pigs
Born 1/16-18/2011 Patent Pending
Prather et al ‘13 J Virology
Prather et al ‘13 J Virology
• Piglets were shipped to Kansas State University
• After 1 week acclimation
– Low passage PRSSV isolate (KS-06: North Ame...
• Assessed daily for clinical signs
– Pigs co-housed
– No apparent clinical signs before infection
– Day 3 after infection...
• Flow Cytometry on porcine alveolar macrophages at Kansas
State University- Bob Rowland.
CD163SIGLEC1Wild-Type
pig
2nd Ab...
CD163SIGLEC1Wild-Type
pig
2nd Ab
SIGLEC1-/-
pig
CD163SIGLEC12nd Ab
Prather et al ‘13 J Virology
Prather et al ‘13 J Virology
SIGLEC1
• Virus in blood that killed cells in vitro- cytopathic
effect.
Prather et al ‘13 J Virology
• Viral RNA/nuclei acid
Prather et al ‘13 J Virology
• Conceive Idea – June ‘02.
• Search for $$$
• Founder male heterozygotes – Born Jan ‘11.
• Founders reach puberty Oct-Sep...
• SIGLEC1-/- is not embryonic lethal.
• Potential problem- KO mice have subtle changes in T- and B-
cell populations, and ...
• A series of experiments similar to that for SIGLEC1
provided evidence that CD163 is not only an entry
mediator but also ...
CD163 is a member of the scavenger
receptor cysteine-rich (SRCR) superfamily
17 exons, 16 introns
• Determine which extracellular domains
were required for PRRSV infectivity
• HEK293T were transfected with SIGLEC1
and CD...
• CD163 is a member of gene family
– hCD163L (CD163-like)
– 12 extracellular domains
– Does not function in infectivity
• ...
Van Gorp et al
2010 J Vir
Human CD163
Human CD163L
• Traditional knockout by homologous recombination and
addition of a stop codon
• Domain Swap
– Remove extracellular domai...
• Haptoglobin-Hemaglobin – SRCR #3
• Erythroblast – SRCR #2
• TNFSF12 (aka TWEAK) – SRCR #1-4 & #6-9
• Bacteria – SRCR #2
...
• Haptoglobin’s ability to promote binding of hemoglobin to
CD163 in human has high affinity, but not in mice.
• In mice C...
• Non-Homologous End Joining
• Can result in changing a handful of base pairs
• Thus can remove exons/domains or knockout ...
• Genomic Sequence Confirmed and CRISPRs
designed (1 day, if good EST data available)
– 7-18-13
• CRISPRs pairs ordered fr...
• Engineering Somatic Cells (1 month to 2 years)
• SCNT/ Zygote injection (1 week)
• Gestation (3 months, 3 weeks & 3 days...
Weaned
wildtype
and CD163
edited
piglets
prior to
transport to
Kansas
State
University.
Patent Pending
• 3 piglets predicted to be null (CD163-/- )
• 8 WT piglets
– One WT piglet humanely euthanized on day 1, due to poor body...
• Identity blinded to the crew at KSU
• Housed in the K-State BL-2 LARC facility
• Allowed to acclimate for 3 days after a...
http://www.scuba-diving-
smiles.com/palm-tree-clipart.html
• Fever was considered positive if it was ≥ 104°F
• Respiratory scores ranged from:
– 0: Normal, to
– 1: mild dyspnea and/...
Whitworth et al.
• Surface expression of CD163 or CD169 on porcine
alveolar macrophages (PAMs) was measured using
flow cytometry
• PRRSV vi...
Whitworth et al.
• CD163-/- pigs had interstitial edema with the infiltration of
mononuclear cells and the mononuclear infiltrate consisted...
Pig Genotype Description Score
*
41 Wild Type 100% congestion. Multifocal areas of edema. Infiltration of moderate
numbers...
Whitworth et al
• SIGLEC1-/- pigs are not resistant to PRRSv.
• CD163 -/- pigs are resistant to PRRSv.
• Working on challenging other geno...
Heparan Sulphate
Binding
Binding and
Internalization
by
Sialoadhesin
Uncoating of
the virus by
CD163
Genome Release
in end...
• Answer Basic and Applied Questions
about Human Medicine and Domestic
Animal Biology
Zygote Injections
Kiho Lee
Lee Spate
CRISPR and Targeting Vector Design
Kevin Wells
Kristin Whitworth
Transfection and Gen...
• 10th International Conference on Pig Reproduction
June 11-14, 2017, University of Missouri, Columbia,
MO
• Open Postdoc ...
Dr. Randy Prather - Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs
Dr. Randy Prather - Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs
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Dr. Randy Prather - Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs

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Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs - Dr. Randy Prather, University of Missouri, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.

More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium

Published in: Science
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Dr. Randy Prather - Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs

  1. 1. • Cancer (CAG-Floxed-Stop, KRASG12D, p53R167H; caCSN2-miBRCA1-RASSF1) • Cardiovascular Disease (hFAT-1+; TEK-NOS3+; TEK-CAT+) • Cardiac Development (Floxed FGF8) • Cystic Fibrosis (CFTR-/-; CFTRΔF508/ΔF508; CFTRΔF508/ΔF508, Tet-on CFTR+; CFTRΔF508/ΔF508, FABP2-CFTR+; shRNA CFTR) • Diabetes (ssGIP-hINS+) • Disease Resistance (SIGLEC1-/-; CD163-/-; CD163-/Δdomain 5; CD163-/Domain 5 swap with domain 8 of hCD163L; CD163-/-, SIGLEC1-/-; CD1D-/-) • Enhancing Meat Quality (hFAT1+) • Muscular Dystrophy (DMD+/Δexon 46) • Pharmaceuticals (FIX+, VWF+, SERPINA1+; FVIII+, SERPINA1+, FURIN+) • Regenerative Medicine (IL2RG+/-; RAG2-/-) • Retinitis Pigmentosa (RHOP23H) • Spinal Muscular Atrophy (SMN+/-; SMN+/-, hSMN2+) • Suicide Prodrug for Liver Transplantation (ALB-TK1+; AFP-CDA+) • Tracking/Tools (CAG-eGFP+; CMV-eGFP+; ZP3-CRE; UBC-Tomato+; CAG-Tomato+; UBC-nls eGFP+; PSMA1-eGFP fusion protein +) • Xenotransplantation (GGTA1-/-; GGTA1-/-, CD55+; GGTA1CD55/+; GGTA1-/-, CD55+, CD59+, ENTPD1+, THBD+; SIGLEC1-/-) • Plus others that I can’t talk about- over 45 different modifications and over 1,000 cloned pigs.
  2. 2. • Model of Infection for PRRSv • SIGLEC1 • CD163 • Timelines & From Here to Where?
  3. 3. – Initial binding with heparan sulfate – Binding/Internalization by Sialoadhesin – Internalization/uncoating of the virus by CD163 Van Breedam et al., 2010
  4. 4. Heparan Sulphate Binding Binding and Internalization by Sialoadhesin (SIGLEC1) Uncoating of the virus by CD163 Genome Release in endosomes dependent on low pH Van Breedam et al., 2010
  5. 5. Southern Probe Prather et al ‘13 J Virology
  6. 6. • A targeted clone (4-18) was identified and used both for somatic cell nuclear transfer and to propagate for Southern blot analysis. • Screened >2,000 colonies: efficiency ~7.0 x 106 Prather et al ‘13 J Virology
  7. 7. Enucleation Fusion or Injection and Activation Development Transfect or Transduce and Select Synchronize? Nuclear Transfer US Patent #6,211,429
  8. 8. SIGLEC1+/- pigs Born 1/16-18/2011 Patent Pending Prather et al ‘13 J Virology
  9. 9. Prather et al ‘13 J Virology
  10. 10. • Piglets were shipped to Kansas State University • After 1 week acclimation – Low passage PRSSV isolate (KS-06: North American isolate collected in 2006) – 105 TCID50 of virus diluted in 3 mL • ½ intramuscularly • ½ intranasally – Blood sampled on days 0, 4, 7, 14, 21, 28 & 35. Prather et al ‘13 J Virology
  11. 11. • Assessed daily for clinical signs – Pigs co-housed – No apparent clinical signs before infection – Day 3 after infection all pigs began to exhibit mild respiratory signs (sneezing, coughing, increased breathing) – These resolved within 1- to 2-weeks. In a single WT pig more severe signs reappeared on day 28. • Euthanized at the end of the study – Histology- Lungs- no difference as all had moderate to severe multifocal interstitial pneumonia. – Collected alveolar macrophages. Prather et al ‘13 J Virology
  12. 12. • Flow Cytometry on porcine alveolar macrophages at Kansas State University- Bob Rowland. CD163SIGLEC1Wild-Type pig 2nd Ab Prather et al ‘13 J Virology
  13. 13. CD163SIGLEC1Wild-Type pig 2nd Ab SIGLEC1-/- pig CD163SIGLEC12nd Ab Prather et al ‘13 J Virology
  14. 14. Prather et al ‘13 J Virology SIGLEC1
  15. 15. • Virus in blood that killed cells in vitro- cytopathic effect. Prather et al ‘13 J Virology
  16. 16. • Viral RNA/nuclei acid Prather et al ‘13 J Virology
  17. 17. • Conceive Idea – June ‘02. • Search for $$$ • Founder male heterozygotes – Born Jan ‘11. • Founders reach puberty Oct-Sept ’11. • F1’s born (heterozygous males and females) Nov ’11-Jan ‘12. • F1’s reach puberty summer ‘12. • Homozygous Knockout animals born Oct ‘12. • Challenge experiments began Nov ‘12. • Publication ’13 almost 11 years later. Prather et al ‘13 J Virology
  18. 18. • SIGLEC1-/- is not embryonic lethal. • Potential problem- KO mice have subtle changes in T- and B- cell populations, and reduced immunoglobulin M (Oetke et al ‘06). • SIGLEC1-/- may have altered IgG response, but variation was high and sample size was small. • SIGLEC1-/- did not affect susceptibility to PRRSv. • Could cells from these pigs be useful diagnostic tools? Prather et al ‘13 J Virology
  19. 19. • A series of experiments similar to that for SIGLEC1 provided evidence that CD163 is not only an entry mediator but also facilitates infection (as outline by Welsh & Calvert ’10). Other candidates include CD151, VIM and CD209.
  20. 20. CD163 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily 17 exons, 16 introns
  21. 21. • Determine which extracellular domains were required for PRRSV infectivity • HEK293T were transfected with SIGLEC1 and CD163 deletion constructs • Cells were inoculated with PRRSv for 24 hr and fixed • Number of infected cells were counted Van Gorp et al 2010 J Vir
  22. 22. • CD163 is a member of gene family – hCD163L (CD163-like) – 12 extracellular domains – Does not function in infectivity • Swap domains from hCD163-like and CD163 and determine infectivity
  23. 23. Van Gorp et al 2010 J Vir Human CD163 Human CD163L
  24. 24. • Traditional knockout by homologous recombination and addition of a stop codon • Domain Swap – Remove extracellular domain SRCR5 from CD163 – Replace with extracellular domain 8 from hCD163L
  25. 25. • Haptoglobin-Hemaglobin – SRCR #3 • Erythroblast – SRCR #2 • TNFSF12 (aka TWEAK) – SRCR #1-4 & #6-9 • Bacteria – SRCR #2 • PRRSv- SRCR #5
  26. 26. • Haptoglobin’s ability to promote binding of hemoglobin to CD163 in human has high affinity, but not in mice. • In mice CD163 accounts for a small part of hemoglobin clearance. • Knockout of CD163 results in “No apparent phenotype change… and offspring were viable and fertile”. Etzerodt et al ‘12. Antioxidants & Redox Signaling, DOI: 10.1089/ars.2012.4605
  27. 27. • Non-Homologous End Joining • Can result in changing a handful of base pairs • Thus can remove exons/domains or knockout a gene. • Swine genome is ~2,700,000,000 base pairs.
  28. 28. • Genomic Sequence Confirmed and CRISPRs designed (1 day, if good EST data available) – 7-18-13 • CRISPRs pairs ordered from IDT (1 day) – 7-19-13 • CRISPRs pairs annealed and ligated and transformed • Bacterial Colonies picked, propagated and sequence confirmed • Positive colonies re-propagated in larger preps for transfections – 7/22/13-7/26/13 (1 week) • Design Assays for smaller and large deletions – This project had large assays – Small assay 435 bp was developed (2 days) • Cells ready for transfection 8-7-14 • First Transfection with CRISPRs (14 days) – 8/9/13-8/23/13 • Colonies picked and genotyped (2 days) – 8/23/14-8/24/14 • Positive Colonies were propagated and sequence confirmed – 8-26-13 (this took along time) • Nuclear Transfer – 9/19/13 • Embryo Transfer – 9/20/13 • First Pigs modified by CRISPRS 1/13/14 Total Time by SCNT: 5.5 - 6 months
  29. 29. • Engineering Somatic Cells (1 month to 2 years) • SCNT/ Zygote injection (1 week) • Gestation (3 months, 3 weeks & 3 days) • Puberty (6-9 months)
  30. 30. Weaned wildtype and CD163 edited piglets prior to transport to Kansas State University. Patent Pending
  31. 31. • 3 piglets predicted to be null (CD163-/- ) • 8 WT piglets – One WT piglet humanely euthanized on day 1, due to poor body condition and not included in the study • K-State veterinarians and staff blinded to genotypes Piglet ID Predicted Translation Maternal Allele Paternal Allele #43 CD163-/- 7 bp addition in exon 7 2 bp deletion in exon 7 + 377 bp intron deletion in the preceding intron #55 CD163-/- 7 bp addition in exon 7 2 bp deletion in exon 7 + 377 bp intron deletion in the preceding intron #40 CD163-/- 7 bp addition in exon 7 11 bp deletion in exon 7
  32. 32. • Identity blinded to the crew at KSU • Housed in the K-State BL-2 LARC facility • Allowed to acclimate for 3 days after arrival at facility • Challenge IM and IN with 105 TCID50 of NVSL 97- 7985. (Standard lab isolate from 1997, relatively “hot” in terms of replication and pathogenesis) • Pigs were maintained in the same pen; therefore, all pigs constantly exposed to virus • Monitored daily for clinical signs • Blood collected on days 0,4,7,11,14,21,28,35 and weights collected at least weekly • Study terminated 35 days after infection • Lung lavage for PAMs • Lungs and tissues removed for histopathology
  33. 33. http://www.scuba-diving- smiles.com/palm-tree-clipart.html
  34. 34. • Fever was considered positive if it was ≥ 104°F • Respiratory scores ranged from: – 0: Normal, to – 1: mild dyspnea and/or tachypnea when stressed (when handled), – 2: mild dyspnea and/or tachypnea when at rest, – 3: moderate dyspnea and/or tachypnea when stressed (when handled), – 4: moderate dyspnea and/or tachypnea when at rest, – 5: severe dyspnea and/or tachypnea when stressed (when handled), – 6: severe dyspnea and/or tachypnea when at rest. Is there evidence of diarrhea (grade) or vomiting?
  35. 35. Whitworth et al.
  36. 36. • Surface expression of CD163 or CD169 on porcine alveolar macrophages (PAMs) was measured using flow cytometry • PRRSV viremia in serum measured using commercial Tetracore assay with standards. Results reported as log10 templates per PCR reaction • PRRSV N protein specific antibody response measured by Luminex. Results reported as S/P ratio S/P = (Sample-BKG)/(Positive-BKG) • Histopathology and scoring of microscopic lesions performed by a board certified anatomic pathologist, Dr. Giselle Cino Whitworth et al.
  37. 37. Whitworth et al.
  38. 38. • CD163-/- pigs had interstitial edema with the infiltration of mononuclear cells and the mononuclear infiltrate consisted mainly of lymphocytes and plasma cells and lesser numbers of macrophages. In contrast there was no evidence for pulmonary changes in the CD163-/- pigs Whitworth et al.
  39. 39. Pig Genotype Description Score * 41 Wild Type 100% congestion. Multifocal areas of edema. Infiltration of moderate numbers of lymphocytes and macrophages. 3 42 Wild Type 100% congestion. Multifocal areas of edema. Infiltration of moderate numbers of lymphocytes and macrophages. 3 47 Wild Type 75% multifocal infiltration with of mononuclear cells and mild edema. 2 50 Wild Type 75% moderate infiltration of mononuclear cells within alveolar spaces and around small blood vessels. Perivascular edema. 3 51 Wild Type 25% atelectasis with moderate infiltration of mononuclear cells. 1 52 Wild Type 10% of alveolar spaces collapsed with infiltration of small numbers of mononuclear cells. 1 56 Wild Type 100% diffuse moderate interstitial infiltration of mononuclear cells. Interalveolar septae moderately thickened by hemorrhage and edema. 4 40 CD163-/- No changes 0 43 CD163-/- No changes 0 55 CD163-/- No changes 0 *Lung lesion scores calculated according to Halbur et al. 8 Vet Pathol. 1995 Nov;32(6):648-60 Whitworth et al
  40. 40. Whitworth et al
  41. 41. • SIGLEC1-/- pigs are not resistant to PRRSv. • CD163 -/- pigs are resistant to PRRSv. • Working on challenging other genotypes. • Working on challenging with other isolates (multiple Type 1 and Type 2).
  42. 42. Heparan Sulphate Binding Binding and Internalization by Sialoadhesin Uncoating of the virus by CD163 Genome Release in endosomes dependent on low pH Van Breedam et al., 2010 CD163 is a Gatekeeper!
  43. 43. • Answer Basic and Applied Questions about Human Medicine and Domestic Animal Biology
  44. 44. Zygote Injections Kiho Lee Lee Spate CRISPR and Targeting Vector Design Kevin Wells Kristin Whitworth Transfection and Genotyping Mykel Anderson Mariah Thomas Joshua Benne Nuclear Transfer and Embryo Transfer Joshua Benne Stephanie Murphy Jennifer Teson Jiude Mao Clifton Murphy Surrogate and Piglet Care Melissa Samuel Jason Dowell Tricia Meyer Entire Prather Lab Funding Sources Genus plc The Christopher Columbus Fellowship Foundation Food for the 21st Century USDA ARS Genus plc Alan Mileham Dave McLaren Jon Lightner SIGLEC1-/- Project Jon Green Tina Egen Kansas State University Bob Rowland Benjamin Trible Maureen Kerrigan Catherine Ewen Ada Cino-Ozuna Bhupinder Bawa
  45. 45. • 10th International Conference on Pig Reproduction June 11-14, 2017, University of Missouri, Columbia, MO • Open Postdoc Position: Molecular Biologist that wants to create additional models. PratherR@Missouri.Edu

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