University of Missouri
Animal Reproductive Biology Group
Prather Laboratory

Genetically Modified/Cloned Pigs/ Assisted Reproductive Technologies at MU

A Timeline

Date

Event

Publication

1989

First cloned pig from an embryonic donor cell
First cloned pig from embryonic donor cell

(Prather et al., 1989)

1995

Our first births resulting from non-surgical embryo transfer
First births resulting from non-surgical embryo transfer

(Li et al., 1996)

February 29, 2000

First transgenic pig from an in vitro system: enhanced green fluorescent protein (eGFP) via oocyte transduction
First transgenic pig from an in vitro system: enhanced green fluorescent protein (eGFP) via oocyte transduction

(Cabot et al., 2001)

March 9, 2001

First cloned transgenic pig: eGFP
First cloned transgenic pig: eGFP

(Park et al., 2001)

September 21, 2001

First knockout pig: alpha 1, 3 galactosyltransferase
First knockout pig: alpha 1, 3 galactosyltransferase

(Lai et al., 2002)

November 18, 2002

First homozygous knockout miniature pig

First homozygous knockout miniature pig

(Kolber-Simonds et al., 2004)

July 29, 2002-2003

Transgenic pigs with a liver specific promoter driving a suicide gene

Transgenic pigs with a liver specific promoter driving a suicide gene

(Beschorner et al., 2003a; Beschorner et al., 2003b)

February 12, 2003

Spot clones born
Spot Clones born

(Carroll et al., 2005)

2003

National Swine Resource and Research Center (NSRRC) FundedNational Swine Resource and Research Center

 

July 6, 2004

hDAF transgenic pigs with alpha 1, 3 galactosyltransferase knockout bornhDAF transgenic pigs with alpha 1, 3 galactosyltransferase knockout born

(Lai et al., Unpublished)

June 24, 2005

Fat-1 transgenic pigs: converts omega 6 fatty acids to omega 3 fatty acids piglets bornFat-1 transgenic pigs: converts omega 6 fatty acids to omega 3 fatty acids piglets born

(Lai et al., 2006)

November 17, 2005

First cryo-preserved transgenic cloned piglets born
First cryo-preserved transgenic cloned piglets born

(Li et al., 2006)

April 12, 2006

pCAG-eGFP transgenic domestic piglets born
pCAG-eGFP transgenic domestic piglets born

(Whitworth et al., 2009)

2006

Completed construction of the NSRRC
National Swine Resource and Research Center

 

May 30, 2006

First CFTR knockout heterozygotes born
First CFTR knockout heterozygotes born

(Rogers et al., 2008a)

July 16, 2006

Clones of pig whose DNA is being sequenced for the Swine Genome Project: TJ TabascoClones of pig whose DNA is being sequenced for the Swine Genome Project: TJ Tabasco

(Hao et al., Unpublished)

June 16, 2007

Our first IVM/IVF/IVC Frozen Blastocysts Piglets Born
Our first IVM/IVF/IVC Frozen Blastocysts Piglets Born

(Li et al., 2009)

July 19, 2007

First Delta F 508 CFTR heterozygotes born
First Delta F 508 CFTR heterozygotes born

(Rogers et al., 2008a)

August 13, 2007

Cloned pigs produced from skin derived stem cells.
Cloned pigs produced from skin derived stem cells.

(Hao et al., 2009)

October 24, 2007

First pigs born as a result in culture in a defined medium with LDL replacing BSA
LDL replace BSA

(Spate et al. 2010)

February 27, 2008

First homozygous CFTR null pig born with CF symptoms
First homozygous CFTR null pig born with CF symptoms

(Rogers et al., 2008b)

March 10, 2008

FVIII, SERPINA1 & VWF tri-transgenic piglets were born
FVIII, SERPINA1 & VWF tri-transgenic piglets

(Zhao et al., Unpublished)

May 26, 2008

P23H Rhodopsin NIH miniature piglets born
P23H Rhodopsin NIH miniature piglets born

(Ross et al., 2012)

June 2, 2008

pCAG-eGFP transgenic Yucatan piglets born
pCAG-eGFP transgenic Yucatan piglets bornpCAG-eGFP transgenic Yucatan piglets born

(Whyte et al., Unpublished)

September 22, 2008

FIX, SERPINA1 & PACE tri-transgenic piglets were born
FIX, SERPINA1 & PACE tri-transgenic piglets were born

(Zhao et al., 2011)

March 16, 2009

Tie2-Catalase/pCAG-eGFP Yucatan piglets born
Tie2-Catalase/pCAG-eGFP Yucatan piglets born

(Whyte et al., 2011b)

November 2, 2009 Tie2-eNOS/pCAG-eGFP Yucatan piglets
(Whyte et al., unpublished)
November 11, 2009

FABP2-CFTR on a CFTR -/- background

(Stoltz et al., 2013).

June 1, 2010

GIP-hINS

(Whyte et al., 2011a)
June 14, 2010

PSMA1-eGFP fusion protein

(Miles et al., 2013)
July 16, 2010

1st step in producing pigs that get spinal muscle atrophy. SMN +/-

(Lorson et al., 2011)
November 8, 2010

Zinc-finger nuclease mediated knockout of eGFP

(Whyte et al. 2011c)

December 13, 2010

GGTA1 +/- with hDAF inserted into GGTA1 – Minnesota miniature pigs

(Beaton et al., 2011)
December 13, 2010

Ossabaw Island clones

(Walters et al., unpublished)

January 16, 2011

SIGLEC1 +/-

(Whitworth et al. 2011)

March 28, 2011

Multi-Xeno pig (GGTA1 -/-, CD55+, CD59+. ENTPD1+, THBD+
asdf

(NSRRC, unpublished).

May 23, 2011

SMN+/-; hSMN2+
asdf

(Lorson et al., unpublished)

January 17, 2012

CAG-NLS-eGFP on the Minnesota Miniature pig background (right piglet)
asdf

(NSRRC, Unpublished)

January 17,2012

CAG-Tomato (dsRed mutant) on the Minnesota Miniature pig background (pig on the left).
asdf

(NSRRC, Unpublished))

February 4, 2012

Zinc-Finger mediated knockout of DMD (exon 46).
asdf

(Ross et al., unpublished)

March 26, 2012

Recreate Ximerex, Inc pig from skin cells. Albumin promoter driving Thymidine Kinase
asdf

(NSRRC, unpublished)

April 9, 2012

Recreate Ximerex, Inc pig from skin cells. Alpha-fetoprotein promoter driving cytosine deaminase
asdf

(NSRRC, unpublished)

April 2, 2012

Zinc-finger mediated knockout of cytidine monophospho-N-Acetylneuraminic acid hydroxylase (CMAH)- heterozygotes
asdf

(Kwon et al., 2013)

April 9, 2012

hDAF into GGTA1 female Minnesota Miniature pigs
asdf

(Beaton et al., unpublished)

May 21, 2012

CAG Lox-Stop, KRAS G12D, p53R167H
asdf

(Schook et al., unpublished)

January 30, 2012

Tet-On CFTR on a CFTR ΔF508/ΔF508 Background
asdf

(Welsh et al., unpublished)

July 9, 2012

Female Ossabaw
asdf

(NSRRC, unpublished)

July 9, 2012

Female Minnesota Miniature pigs with a zona pellucida 3 promoter driving CRE
asdf

(NSRRC, unpublished)

July 23, 2012

ZNF-Mediated Homozygous KO of Cytidine Monophospho-N-Acetylneuraminic Acid Hydroxylase (CMAH- one allele of Homologous Recombination and one of Non-Homologous End Joining)
asdf

(Kwon and others 2013)

September 11, 2012

Ten piglets born from zygotes cultured to blastocyst in ps48
asdf

(Spate et al., unpublished)

October 18, 2012

First SIGLEC1 -/- Pigs (F2 pigs) Born
asdf

(Prather et al., 2013)

February 4, 2013

Pigs with a TALEN-mediated knockout of interleukin 2 receptor gamma (IL2Rγ+/- )
asdf

(Lee et al., unpublished)

February 18, 2013

This Minnesota miniature pig has a ubiquitin C promoter driving “tomato.”
asdf

(NSRRC, unpublished)

March 1, 2013

This Minnesota miniature pig has a ubiquitin C promoter driving a nuclear localized eGFP
asdf

(NSRRC, unpublished)

April 15, 2013

Male pigs that have a mutation in the recombination activating gene 2 were produced (RAG2 Δ140-527/Δ140, S141P and RAG2+/Δ140, S141P) in collaboration with Jin-Hoi Kim. Homozygous animals do not produce mature B or T lymphocytes
asdf

(Lee et al., 2014)

October 1, 2013

A breast cancer model. CSN2 promoter driving micro RNAs directed against BRACA1and RassF1a. In collaboration with Geoff Clark, U. Louisville.
asdf

(NSRRC, unpublished)

13/Feb 4/Mar 10, 2014

CD163 or CD163 and SIGLEC1. Created by using CRISPRs and SCNT or zygote injection.
asdf

(al., BOR, in press))

March 17/April 9, 2014

Pigs with a mutation in CD1D that should eliminate natural killer T-Cells. In collaboration with Dr. Driver, U. FL. Created by using CRISPRs and SCNT or zygote injection.
asdf

(Whitworth et al., BOR in press)

March 31, 2014

Pigs that have both genes knocked out for GGTA1-/- and CMAH -/-. In collaboration with Jin-Hoi Kim at Konkuk University in Korea.
asdf

(Lee et al., 2014)

 

Patents Resulting from Pig Work at Missouri

  1. J. Li, A. Rieke, B. Day & R.S. Prather, "Method of Effecting Embryo Transplant"; U.S. #5,558,636 (issued September 24, ‘96).
  2. J. Li, A. Rieke, B. Day & R.S. Prather, "A System for Effecting Embryo Transplant"; U.S. #5,656,010 (issued August 12, ‘97).
  3. J. Li, A. Rieke, B. Day & R.S. Prather, “A System for Introducing a Fluid into the Uterus of an Animal”; U.S. #5,916,144 (issued June 29, ‘99).
  4. J. Li, A. Rieke, B. Day & R.S. Prather, “A System for Introducing a Fluid into the Uterus of an Animal”; Australia #713345 (issued March 16, ’00).
  5. J. Li, A. Rieke, B. Day & R.S. Prather, “A method for Introducing a Fluid into the Uterus of an Animal”; Australia #717684 (issued July 13, ’00).
  6. Z. Machaty & R.S. Prather, “Complete Activation of Mammalian Oocytes”. U.S. 6,211,429 (issued April 3, 01).
  7. J. Li, A. Rieke, B. Day & R.S. Prather, “A System for Introducing a Fluid into the Uterus of an Animal”; Canada. #2,220,550 (issued April 17, ’01).
  8. Prather, R.S., B.N. Day & R. Hawley. “Knockout swine and methods for making the same” U.S. #7,547,816 (issued June 16, ’09).
  9. Prather, R.S., “Improvement of fertilization and development of in vitro produced embryos”, Provisional Patent filed June 25, ’04.
  10. Welsh, M.J., C. Rogers, R.S. Prather, J. Engelhardt, Z. Yan. “Transgenic Animal Models of Disease”, Provisional patent filed March 30, ’07, Utility Patent Application filed March 5, ‘08.
  11. Prather, R.S., “Improvement of fertilization and development of in vitro produced embryos”, Provisional Patent filed June 25, ’04.
  12. Li, R., L. Spate, C.N. Murphy, R.S. Prather. “High throughput cryopreservation of swine embryos”, Provisional Patent filed August 29, ’08. US61/190,515.
  13. Prather, R.S. “Porcine reproductive and respiratory syndrome virus resistant animals”, Provisional Patent filed May 16, ’11.

 

References

Beaton, B.P., Walters, E.M., Prather, R.S., and Wells, K.D. (2011). Alpha-Galactosyltransferase (α1,3-Gal) Knockout Genetically Linked to Xenograft Transgenes. Swine in Biomedical Research Conference July 17-19, Chicago, IL., 28.

Beschorner, W., Joshi, S.S., Prather, R., Schieber, T., Thompson, S.C., Yang, T., Zhou, L., and Mirvish, S. (2003). Selective and conditional depletion of pig cells with transgenic pigs and specific liposomes. Xenotransplantation 10, 497.

Cabot, R.A., Kuhholzer, B., Chan, A.W.S., Lai, L., Park, K.W., Chong, K.Y., Schatten, G., Murphy, C.N., Abeydeera, L.R., Day, B.N., and Prather, R.S. (2001). Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector. Animal Biotechnology 12, 205-214.

Carroll, J.A., Carter, D.B., Korte, S.W., and Prather, R.S. (2005). Evaluation of the acute phase response in cloned pigs following a lipopolysaccharide challenge. Domestic Animal Endocrinology 29, 564-572.

Hao, Y., Wax, D., Zhong, Z., Murphy, C., Ross, J.W., Rieke, A., Samuel, M., Spate, L., Dyce, P., Li, J., Sutovsky, P., and Prather, R.S. (2009). Porcine skin-derived stem cells can serve as donor cells for nuclear transfer. Cloning & Stem Cells 11, 101-10.

Kolber-Simonds, D., Lai, L., Watt, S.R., Denaro, M., Arn, S., Augenstein, M.L., Betthauser, J., Carter, D.B., Greenstein, J.L., Hao, Y., Im, G.-S., Liu, Z., Mell, G.D., Murphy, C.N., Park, K.-W., Rieke, A., Ryan, D., Sachs, D.H., Forsberg, E.J., Prather, R.S., and Hawley, R.J. (2004). Alpha-1,3-galactosyltransferase null pigs via nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proceedings of the National Academy of Sciences of the United States of America 101, 7335-7340.

Kwon, D.-N., Lee, K., Kang, M.-J., Whyte, J.J., Brown, A.N., Kim, J.-H., Samuel, M., Mao, J., Park, K.W., Murphy, C.N., Prather, R.S., and Kim, J.-H. (2013). Production of CMP-N-acetylneuraminic acid hydroxylase knock-out pigs using zinc finger nucleases, for pig-human xenotransplantation. . Scientific Reports (submitted).

Lai, L., Kang, J.X., Li, R., Wang, J., Witt, W., Yong, H.Y., Hao, Y., Wax, D., Murphy, C.N., Rieke, A., Samuel, M., Linville, M.L., Korte, S.W., Evans, R., Starzl, T.E., Prather, R.S., and Dai, Y. (2006). Generation of cloned transgenic transgenic pigs rich in omega-3 fatty acids. Nature Biotechnology 24, 435-437.

Lai, L.X., Kolber-Simonds, D., Park, K.W., Cheong, H.T., Greenstein, J.L., Im, G.S., Samuel, M., Bonk, A., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B., Hawley, R.J., and Prather, R.S. (2002). Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089-1092.

Lee, K., Kwon, D.N., Ezashi, T., Choi, Y.J., Park, C., Ericsson, A.C., Brown, A.N., Samuel, M.S., Park, K.W., Walters, E.M., Kim, D.Y., Kim, J.H., Franklin, C.L., Murphy, C.N., Roberts, R.M., Prather, R.S., and Kim, J.H. (2014). Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency. Proc Natl Acad Sci U S A 111, 7260-5.

Li, J., Rieke, A., Day, B.N., and Prather, R.S. (1996). Porcine Non-Surgical Embryo Transfer. Journal of Animal Science 74, 2263-2268.

Li, R., Lai, L., Wax, D., Hao, Y., Murphy, C.N., Rieke, A., Samuel, M., Linville, M.L., Korte, S.W., Evans, R.W., Turk, J.R., Kang, J.X., Witt, W.T., Dai, Y., and Prather, R.S. (2006). Cloned transgenic swine via in vitro production and cryopreservation. Biology of Reproduction 75, 226-30.

Li, R., Murphy, C.N., Spate, L., Wax, D., Isom, C., Rieke, A., Walters, E.M., Samuel, M., and Prather, R.S. (2009). Production of piglets after cryopreservation of embryos using a centrifugation-based method for delipation without micromanipulation. Biology of Reproduction 80, 563-71.

Lorson, M.A., Spate, L.D., Samuel, M.S., Murphy, C.N., Lorson, C.L., Prather, R.S., and Wells, K.D. (2011). Disruption of the Survival Motor Neuron (SMN) gene in pigs using ssDNA. Transgenic Res.

Miles, E.L., O'Gorman, C., Zhao, J., Samuel, M., Walters, E., Yi, Y.J., Sutovsky, M., Prather, R.S., Wells, K.D., and Sutovsky, P. (2013). Transgenic pig carrying green fluorescent proteasomes. Proc Natl Acad Sci U S A 110, 6334-9.

Park, K.W., Cheong, H.T., Lai, L.X., Im, G.S., Kuhholzer, B., Bonk, A., Samuel, M., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B., and Prather, R.S. (2001). Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein. Animal Biotechnology 12, 173-181.

Prather, R.S., Rowland, R.R., Ewen, C., Trible, B., Kerrigan, M., Bawa, B., Teson, J.M., Mao, J., Lee, K., Samuel, M.S., Whitworth, K.M., Murphy, C.N., Egen, T., and Green, J.A. (2013). An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus. J Virol 87, 9538-46.

Prather, R.S., Sims, M.M., and First, N.L. (1989). Nuclear transplantation in early pig embryos. Biology of Reproduction 41, 414-418.

Rogers, C.S., Hao, Y., Roklina, T., Samuel, M., Stoltz, D.A., Li, Y., Petroff, E., Vermeer, D.W., Kabel, A.C., Yan, Z., Spate, L., Wax, D., Murphy, C.N., Rieke, A., Whitworth, K., Linville, M.L., Korte, S.W., Engelhardt, J.F., Welsh, M.J., and Prather, R.S. (2008a). Production of CFTR-null and CFTR-deltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer. Journal of Clinical Investigation 118, 1571-1577.

Rogers, C.S., Stoltz, D.A., Meyerholz, D.K., Ostedgaard, L.S., Rokhlina, T., Taft, P., Rogan, M.P., Pezzulo, A.A., Karp, P.H., Itani, O.A., Kabel, A.C., Wohlford-Lenane, C.L., Davis, G.J., Smith, T.L., Samuel, M., Wax, D., Murphy, C.N., Rieke, A., Whitworth, K., Uc, A., Starner, T.D., Brogden, K.A., Shilyansky, J., McCray, P., Jr., Zabner, J., Prather, R.S., and Welsh, M.J. (2008b). Disruption of the CFTR gene produces a model of cystic fibrosis in newborn piglets. Science 321, 1837-1841.

Ross, J.W., Castro, J.F., Zhao, J., Samuel, M., Walters, E.M., Rios, C., Bray-Ward, P., Jones, B.W., Marc, R.E., Wang, W., Zhou, L., Noel, J.M., McCall, M., DeMarco, P., Prather, R.S., and Kaplan, H.J. (2012). Generation of an inbred miniature pig model of retinitis pigmentosa. Investigative Ophthalmology & Visual Sciences in press.

Spate, L.D., Whitworth, K.M., Walker, K.A., Bauer, B.K., Murphy, C.N., and Prather, R.S. (2010). Low-density lipoprotein (LDL) receptor mRNA and protein may enable LDL to replace bovine serum albumin during the in vitro swine embryo development. Molecular Reproduction & Development 77, 298.

Stoltz, D.A., Rokhlina, T., Ernst, S.E., Pezzulo, A.A., Ostedgaard, L.S., Karp, P.H., Samuel, M.S., Reznikov, L.R., Rector, M.V., Gansemer, N.D., Bouzek, D.C., Alaiwa, M.H., Hoegger, M.J., Ludwig, P.S., Taft, P.J., Wallen, T.J., Wohlford-Lenane, C., McMenimen, J.D., Chen, J.H., Bogan, K.L., Adam, R.J., Hornick, E.E., Nelson, G.A.t., Hoffman, E.A., Chang, E.H., Zabner, J., McCray, P.B., Jr., Prather, R.S., Meyerholz, D.K., and Welsh, M.J. (2013). Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs. J Clin Invest 123, 2685-93.

Whitworth, K., Egan, T., Teson, J., Green, J.A., Wells, K.D., and Prather, R.S. (2011). Disruption the Sialoadhesin and CD163 Genes to Create Pigs Resistant to PRRSV Infectivity. Swine in Biomedical Research Conference July 17-19, Chicago, IL., 39.

Whitworth, K.M., Li, R., Spate, L.D., Wax, D.M., Rieke, A., Whyte, J.J., Manandhar, G., Sutovsky, M., Green, J.A., Sutovsky, P., and Prather, R.S. (2009). Method of oocyte activation affects cloning efficiency in pigs. Mol Reprod Dev 76, 490-500.

Whyte, J.J., Isom, S.C., Samuel, M., Asadi, A., Kieffer, T.J., and Prather, R.S. (2011a). Human insulin expression under control of the Sus scrofa proximal GIP promoter in intestinal K cells of transgenic swine. Swine in Biomedical Research Conference July 17-19, Chicago, IL., 41.

Whyte, J.J., Samuel, M., Mahan, E., Padilla, J., Simmons, G.H., Arce-Esquivel, A.A., Bender, S.B., Whitworth, K.M., Hao, Y.H., Murphy, C.N., Walters, E.M., Prather, R.S., and Laughlin, M.H. (2011b). Vascular endothelium-specific overexpression of human catalase in cloned pigs. Transgenic Res 20, 989-1001.

Whyte, J.J., Zhao, J., Wells, K.D., Samuel, M.S., Whitworth, K.M., Walters, E.M., Laughlin, M.H., and Prather, R.S. (2011c). Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs. Mol Reprod Dev 78, 2.

Zhao, J.G., Walters, E.M., Calcaterra, J., Ross, J.W., Spate, L.D., Samuel, M.S., Rieke, A., Murphy, C.N., Butler, S.P., Velander, W.H., and Prather, R.S. (2011). Bioengineering of the mammary gland of livestock: increased propeptide processing of Factor IX in the milk of transgenic pigs by co-expressing furin. Reproduction, Fertility, & Development 23, 339.