Progress 01/01/15 to 12/31/22
Outputs
Target Audience:Results from the research will be disseminated to the scientific community, students and academicians at several national and international meetings acknowledging the funding source Changes/Problems:Generating a cohort of animals with pre-determined modification via SCNT has proven challenging during this study. Keeping this into consideration, we will pivot to zygotic injections and embryo transfers for generating G0 animals and subsequent breeding to generate the number of animals required for challenge studies and investigating gene function in the future. What opportunities for training and professional development has the project provided?A postdoctoral fellow was trained on generating the knockout and knockin fibroblast cells and generating live animals by somatic cell nuclear transfer. A graduate student was trained on performing IAV challenge studies and histological analysis of lungs, Virologic assays and PCR antigen detection, and Pathology and Immunohistochemistry staining. How have the results been disseminated to communities of interest?We are finalizing the manuscript for publication in a peer-reviewed journal. We will disseminate the results of these studies via the publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We performed 4 embryo transfers of the ST6GAL1 knockout / MX1 knockin fibroblast in the month of August. Two of them were pregnant at day 60 and only one of the two sows farrowed and delivered 5 live piglets. However, the piglets were less thrifty and piglets were either dead or were had to be humanely euthanized. It is clear based on our multiple efforts and repeated failures to generate a cohort of ST6GAL1 knockouts that the ST6GAL1 mutations are not cloneable, and/ or the mutations are making the animals less thrifty. Based on these findings, we have changed the target for genetic modification to TMPRSS2, a serine protease essential for virus infectivity and spread. The protease is required for cleaving hemagglutinin and release of the virus in the endosomes, and subsequent required for downstream replication and infectivity of the IAV. As such this is an attractive candidate for genetic modification. We have generated a total of 7 TMPRSS2-/- piglets and shipped to our Collaborator Dr. Amy Vincent for Flu challenge studies. The pigs were separated into two groups and housed in separate containment rooms, one group of positive control wildtype pigs (N=10) and a group of TMPRSS2 knock-out gene (KO) pigs (N=7). After arrival, the pigs were housed in bio-safety level 2 containment and cared for in compliance with the Institutional Animal Care and Use Committee of the National Animal Disease Center. The animals were treated with ceftiofur crystalline free acid and tulathromycin (Zoetis Animal Health, Florham Park, NJ) to reduce secondary bacterial infections. All were screened by a commercial ELISA kit to ensure absence of IAV (Swine Influenza Virus Antibody Test, IDEXX, Westbrook, ME). At 4-5 weeks of age, pigs were inoculated intratracheally and intranasally with 2 ml and 1 ml, respectively, of 1 x 10^5 50% tissue culture infectious dose (TCID50)/ ml of the IA/20 virus. Inoculation was done under anesthesia, using an intramuscular injection of ketamine (8 mg/kg of body weight; Phoenix, St. Joseph, MO), xylazine (4 mg/kg; Lloyd Inc., Shenandoah, IA), and Telazol (6 mg/kg; Zoetis Animal Health, Florham Park, NJ) cocktail. Nasal swabs (NS) were collected daily from 0 to 5 days post-inoculation (dpi), in infection media containing 2 ml minimum-essential-medium (MEM) supplemented with 1:1000 TPCK Trypsin, to evaluate nasal viral shedding. On 5 dpi the pigs were bled and humanely euthanized with a lethal dose of pentobarbital (Fatal Plus; Vortech Pharmaceuticals, Dearborn, MI). Lungs were aseptically removed, evaluated for macroscopic lesions and lavaged with 50 ml of MEM containing 1% bovine serum albumin (BSA) to obtain bronchoalveolar lavage fluids (BALF). Right cardiac or affected lung lobe and distal trachea tissues were collected from the positive control pigs. The TMPRSS2- KO pigs had additional nasal turbinate, kidney, spleen, testicular and rectal tissues collected and fixed in 10% buffered formalin for histopathologic examination, scoring and staining. Virologic assays and PCR antigen detection: All nasal swab samples were filtered with a 0.2 micron syringe filter and plated onto 48-well plates containing confluent MDCK cells that were washed twice with phosphate-buffered saline (PBS) and cultured at 37? for 48 to 72 hours, depending on the amount of cytopathic effect present. Plates were fixed and stained for immunocytochemistry (ICC) as previously described (Arendsee et al. Microbiol Resour Announc. 2021 Dec 16;10(50):e0108121. doi: 10.1128/MRA.01081-21). Virus isolation-positive nasal swabs and BALF samples were analyzed for viral titers by being plated onto 96-well plates of MDCK cells in 100µl 10-fold serial triplicate dilutions, with infection media containing antibiotics. At 48 hours, plates were fixed and stained. Titers were calculated for each sample as TCID50 per ml and transformed to log10 for comparison purposes. Nasal swab samples were submitted to RNA isolation by MagMAX-96 Viral RNA Isolation Kit and qPCR thru VetMAXTM-Gold SIV Detection Kit (Thermo Fisher Scientific, Waltham, MA) to compare cycle threshold (CT) values. Pathology and Immunohistochemistry staining At necropsy, the percentages of lung surface that contained lesions typical of IAV infection were recorded. A visual estimation was made for each lung lobe, and a total percentage for the entire lung was calculated based on weighted proportions of each lobe to the total lung volume (Halbur et al.. 1995. Vet Pathol 32:648-660. https://doi.org/10.1177/030098589503200606). All tissues collected and fixed in 10% formalin were transferred to 70% ethanol buffer approximately 48 hours following collection in formalin. Lung and tracheal tissues were processed by routine histopathologic procedures and the slides were stained with hematoxylin and eosin. Microscopic lesions were evaluated and scored by a veterinary pathologist by parameters previously described (Gauger et al. 2012.Vet Pathol 49:900 -912. https://doi.org/10.1177/0300985812439724). Selected tissues from wildtype and TMPRSS2- KO pigs underwent routine processing to be used in immunohistochemistry staining essays to detect IAV antigen and confirm the absence of TMPRSS2 expression. Results: Viral replication and shedding No IAV was detected in nasal secretions of any pigs at 0 dpi. Both groups had onset of viral shedding at 1 dpi with 10 of 10 positive in the wildtype control group and 2 of 7 in the TMPRSS2- KO group. However, all 7 pigs in the - KO group were shedding on 2 dpi. Viral shedding in nasal swabs continued in both groups throughout the duration of the study. Titers were significantly different between groups on 3 dpi, when the KO pig group had higher (p<0.005) mean titers in nasal swabs than the wild type positive control group. Viral RNA levels were also higher for the KO pigs on dpi 2, reflected in significantly lower CT values (p<0.05). Influenza A virus was detected in BALF samples of all challenged pigs and mean viral titers did not have a significant difference between groups. Lung and tracheal lesions The IA/20 H1N1 isolate induced mild percentages of pneumonia in all pigs. Lungs had varying degrees of multifocal to coalescing areas of consolidation, consistent with experimental infection of IAV. Macroscopic lung lesions demonstrated higher average percentages in the wild type positive control group compared to the TMPRSS2- KO group. Overall, microscopic lung lesions scores followed the same pattern, with the mean scores being significantly lower in the TMPRSS2- KO group. Tracheal lesions were not significantly different between groups. In summary, although we have not witnessed a decline in infectivity of AAV in the TMPRSS2 knockout pigs, we have seen significant improvement in macroscopic and microscopic lung lesions in the knockout pigs compared to the wild type pigs.
Publications
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Progress 01/01/21 to 12/31/21
Outputs
Target Audience:Results from the research were presented to the scientific community, students and academicians at several national and international meetings acknowledging the funding source. Changes/Problems:The COVID outbreakreally affected our ability to perform research. The requirement for social distancing, chronic understaffing and loss of key personnel at the research farm caused a significant delayfor the embryotransfers of the ST6GAL1 knockout and MX1 knockin fibroblast (August 2021). However, with the no-cost extension we will be able to complete the studies as planned What opportunities for training and professional development has the project provided?A postdoctoral fellow was trained on generating the knockout and knockin fibroblast cells and generating live animals by somatic cell nuclear transfer. How have the results been disseminated to communities of interest?We discussed the results in the NIFA PD meeting What do you plan to do during the next reporting period to accomplish the goals?The COVID outbreakreally affected our ability to perform research. The requirement for social distancing, chronic understaffing and loss of key personnel at the research farm had a significant negative impact on our ability to perform embryotransfers of the ST6GAL1 knockout and MX1 knockin fibroblast. We typically avoid embryo transfers in pigs insummer, so we performed the embryo transfers at our earliest available opportunity, which was August. Thepregnant pig is scheduled to farrow December 2021. The co-investigator (Dr. Vincent) intends topurchase pigs and reagents for challenge studies. The granted,a no-cost extension will allow us to complete the challenge study aswas proposed in the grant.
Impacts
What was accomplished under these goals?
We performed 4 embryo transfers of the ST6GAL1 knockout / MX1 knockin fibroblast in the month of August. Two of them are pregnant ard are due to farrow in the first week of December 2021. From ultrasounds, we expect to have 6-8 livepiglets on the ground, which will be shipped to ourCollaborator Dr. Amy Vincent for Flu challenge studies.
Publications
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Progress 01/01/20 to 12/31/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Much of the laboratory work and transfer procedures were stopped due to COVID-19 closures of facilities. What opportunities for training and professional development has the project provided?A postdoc was trained on performing genetic modification in pig fetal fibroblast cells How have the results been disseminated to communities of interest?We discussed the results in the NIFA PD meeting What do you plan to do during the next reporting period to accomplish the goals?We will attempt to perform embryo transfers with the knockout/knockin cells
Impacts
What was accomplished under these goals?
Aim-1: The shRNA approach was not successful. Co-investigators at Roslin attempted to make pig transgenic with shRNA for IAV, but no detectable expression of shRNA was observed Aim-2: We performed embryo injections to generate ST6GAL1/ST3GAL1 double knockout pigs. From a round of embryo transfer we obtained fetuses at day 45 and identified fetuses with a range of mutations. We used one of the ST6GAL1 knockout fetus to introduce an interferon inducible MX transgene .
Publications
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Progress 01/01/19 to 12/31/19
Outputs
Target Audience:Results from the research were presented to the scientific community, students and academicians at several national and international meetings acknowledging the funding source. Changes/Problems: The defective interfering particles/decoys that were proposed in the proposal and since been tested at the Roslin Institute and have been found not be adequately expressed and/or ineffective in mounting resilience against viral infections. In this regard, we have altered the strategy and have since used interferon inducible MX transgene as a second layer of defense, in addition to the receptor ablation. What opportunities for training and professional development has the project provided?A postdoctoral fellow was trained on generating the knockout and knockin fibroblast cells and generating live animals by somatic cell nuclear transfer. How have the results been disseminated to communities of interest?The data was presented at various national and international meetings: 1. University of Missouri- Columbia, 2019 Title: Unlocking the translational potential of pig models 2. Center for Tropical Livestock Genetics and Health Annual Meeting, Edinburgh, UK, 2019 Title: Proof-of-concept (POC) for genome editing in livestock genetic multiplication systems 3. MedCHi Physician Society, Baltimore, MD, 2019 Title: Genome editing in Agricultural aniamls: Opportunities and Challenges. 4. Invited seminar: Department of Veterinary Pathology, College Park, MD, 2019 Title: Genome editing in pigs to develop models of disease 5. 3rd Annual Genome Editing USA Congress, Boston, MA, 2019 Title: Unlocking the translational potential of pig models using genome editors What do you plan to do during the next reporting period to accomplish the goals?The ST6GAL1 null/ IFN: MX transgenic fibroblasts will be utilized for generating pigs for challenge studies. The pigs will be generated by cloning and the piglets shipped to NADC, IA for challenge studies. The phenotype from the resulting animals (resilience or lack therof) will be throughly characterized and the data disseminated to the scientific community
Impacts
What was accomplished under these goals?
The ST6GAL1 knockout pigs were generated from the knockout fibroblasts. The knockout pigs were alive showing the knockouts are not embryonic lethal The fibroblasts are expanded and an interferon inducible MX transgene was stably knocked in.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
1. Rexroad et al., Genome to Phenome: Improving Animal Health, Production, and Well-Being A New USDA Blueprint for Animal Genome Research 20182027.
Frontiers in Genetics. 2019. May 16;10:327. doi: 10.3389/fgene.2019.00327.
2. Telugu BP*, Park KE#, Park CH. Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications. Mammalian Genome. 2017 Jul 15. doi: 10.1007/s00335-017-9709-4. [Epub ahead of print] PMID: 28712062
3. Sheets TP, Park KE, Park CH, Swift SM, Powell A, Donovan DM, Telugu BP*. CRISPR/Cas9 Ablation of NEUROGENIN 3 (NGN3) in Domestic Pigs Impairs Pancreatic Endocrine but not Exocrine Development. Scientific Reports. 2018 Feb 26;8(1):3582. PMID: 29483633.
4. Zhou Y, Shen B, Jiang J, Padhi A, Park KE, Oswalt A, Sattler CG, Telugu BP, Chen H, Cole JB, Liu GE, Ma L. Construction of PRDM9 allele-specific recombination maps in cattle using large-scale pedigree analysis and genome-wide single sperm genomics. DNA Research. 2017 Nov 27. doi: 10.1093. PMID: 29186399
5. Park KE#, Powell A, Sandmaier SES#, Kim C#, Mileham A, Donovan DM, Telugu BP*. Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes. Scientific Reports. 2017 Feb 14; 7:42458. PMID: 28195163
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Progress 01/01/18 to 12/31/18
Outputs
Target Audience:Results from the research were presented to the scientific community, students and academicians at several national and international meetings acknowledging the funding source. Changes/Problems:The defective interfering particles/decoys that were proposed in the proposal and since been tested at the Roslin Institute and have been found not be adequately expressed and/or ineffective in mounting resilience against viral infections. In this regard, we have altered the strategy and have since used interferon inducible MX transgene as a second layer of defense, in addition to the receptor ablation. What opportunities for training and professional development has the project provided?A postdoctoral student and a graduate student have received training in generating targeting constructs, nucleofection and stable selection of cells and generating stable selected cells. How have the results been disseminated to communities of interest?The results have been presented at various national and international conferences (E.g. LAGE, Large Animal Genome Editing Conference) to target audiences. What do you plan to do during the next reporting period to accomplish the goals?Theknockout/knockin cells were used in somatic cell nuclear transfer/cloning to generate a pregnancy. Cell lines from the piglets will be tested for IAV resilience. The validated lines will be used to generate a cohort of pigs for whole animal viral challenge studies to be performed by co-PI, Dr. Amy Vincent at NADC, Iowa.
Impacts
What was accomplished under these goals?
CRISPR/Cas knockout of ST3 and ST6GAL1 knockout animals have been generated. Cell lines from the tracheal epithelium were established for invitro viral challenge experiments. A mouse MX transgene under the regulation of IFNB promoter has been knockin into the ST6GAL1 knockout fibroblasts.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
1) Sheets TP, Park KE, Park CH, Swift SM, Powell A, Donovan DM, Telugu BP*. CRISPR/Cas9 Ablation of NEUROGENIN 3 (NGN3) in Domestic Pigs Impairs Pancreatic Endocrine but not Exocrine Development. Scientific Reports. 2018 Feb 26;8(1):3582. PMID: 29483633.
2) Zhou Y, Shen B, Jiang J, Padhi A, Park KE, Oswalt A, Sattler CG, Telugu BP, Chen H, Cole JB, Liu GE, Ma L. Construction of PRDM9 allele-specific recombination maps in cattle using large-scale pedigree analysis and genome-wide single sperm genomics. DNA Research. 2017 Nov 27. doi: 10.1093. PMID: 29186399
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Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Target audiences reached by the project include academians, researchers, and animal husbandry industry. The target audiences are reached by laboratory instruction, or practicum experiences; development of innovative research methodologies; workshops; and experiential learning opportunities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training activities: The grant serves to train a postdoctoral fellow, a graduate student and a technician in advanced CRISPR/Cas genome editing tools. Professional development: The technical knowledge and skill level of the trainees was enhanced by encouraging participation in workshops, conferences, seminars, study groups, and individual study. How have the results been disseminated to communities of interest?The results were communicated by research articles, review articles and at seminars at national and international meetings (Large animal genetic engineering summing; CRISPR AG Bio conference; 6th Swine in Biomedical Research Conference). What do you plan to do during the next reporting period to accomplish the goals?We plan to validate that the ablation of ST3 and ST6GAL1 results in reduced infection from IAV. Once confirmed, the fibroblast cells will be used for cloning to generate a cohort of animals for challenge studies at NADC, IA.
Impacts
What was accomplished under these goals?
Aim-1: The Roslin collaborators are assembling and validating decoy vectors. Aim-2: We have successfully generated ST3- and ST6-GAL1 double knockout pigs. This illustrates that the double ablation is not embryonic lethal. Primary tracheal and fetal fibroblast cells were established from the pigs. Co-investigators are currently testing the loss of infection in these lines. Aim-3: The fetal fibroblasts from ST3- and ST6GAL1 double knockout pigs will be utilized for the generation of a cohort of pigs for challenge studies in 2018 at NADC, IA
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Park KE, Powell A, Sandmaier SES, Kim C, Mileham A, Donovan DM, Telugu BP. Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes. Scientific Reports. 2017 Feb 14; 7:42458. PMID: 28195163
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Genovese NJ, Domeier TL, Telugu BP, Roberts RM. Enhanced Development of Skeletal Myotubes from Porcine Induced Pluripotent Stem Cells. Scientific Reports. 2017 Feb 6; 7:41833. PMID: 28165492
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Park KE, Kaucher A, Powell A, Waqas SM, Sandmaier SES, Oatley MJ, Park CH, Tibary A, Donovan DM, Blomberg L, Lillico S, Whitelaw CBA, Mileham A, Telugu BP, and Jon M. Oatley (2016). Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Scientific Reports. 2017 Jan 10; 7:40176. PMID: 28071690
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Sheets TP, Park CH, Park KE, Powell A, Donovan DM, Telugu BP. Somatic cell nuclear transfer followed by CRIPSR/Cas9 microinjection results in highly efficient genome editing in cloned pigs. International Journal of Molecular Sciences. 2016 Dec 3;17(12). pii: E2031. PMID: 27918485
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Telugu BP, Park KE, Park CH. Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications. Mammalian Genome. 2017 Jul 15. doi: 10.1007/s00335-017-9709-4. [Epub ahead of print] PMID: 28712062
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:During this funding period, the PI has attended and presented at the Large animal genetic engineering summit, Annual Animal Health meeting in Greensboro, and US-EU meeting on Genome editing in Budapest, Hungary.Using these media, the PI has reachedout to academians, students, and Governmental regulators. Additionally, the PI's work has been highlighted in NY times, You tube, and presented at the National Academy of Medicine.Via these media, the PI has reached out tolay persons. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided opportunity to train a graduate student and a postdoctoral trainee in performing genome editing in livestock. How have the results been disseminated to communities of interest?Yes, the data has been disseminated as publications. What do you plan to do during the next reporting period to accomplish the goals?The ST6- and ST3GAL1 fetal fibroblast cells will be used to generate clonal pigss for viral challenge experiments
Impacts
What was accomplished under these goals?
We have successfully generated and valided the CRISPRs to generate simultaneous knockout of ST6- and ST3GAL1 loci by injection of CRISPR reagents into the cytoplasm of 1-cell pig embryos. We have established a successful pregnancy and collected fetuses for establishing fetal fibroblast lines. We have screened and confirmed double knockout of the ST6- and ST3 GAL1 loci.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
1. Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs.
Sheets TP, Park CH, Park KE, Powell A, Donovan DM, Telugu BP.
Int J Mol Sci. 2016 Dec 3;17(12). pii: E2031.
PMID: 27918485 Free Article
2. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.
Park KE, Park CH, Powell A, Martin J, Donovan DM, Telugu BP.
Int J Mol Sci. 2016 May 26;17(6). pii: E810. doi: 10.3390/ijms17060810.
PMID: 27240344 Free PMC Article
3.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene
Park KE, Kaucher A, Powell A, Waqas MS, Sandmaier SE, Oatley MJ, Park CH,Tibary A, Donovan DM, Blomberg L, Lillico S, Whitelaw CBA, Mileham A, Telugu BP, and Oatley JM
Scientific Reports (Accepted)
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Progress 01/01/15 to 12/31/15
Outputs
Target Audience:Target audience are scientific community and industry groups, primarily animal genetics companies and pharamaceutical companies. These target audience are reached via publication of a manuscript and through News article published in New York Times. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training for a postdoctoral fellow to generate and validate editors for genome editing. The project also allowed the postdoctoral fellow and a technician to generate and validate a landing site into a safe harbor locus. How have the results been disseminated to communities of interest?Yes. A publication to this effect has already been published. Additionally, the PD is already engaged the public into the work that is underway to generate flu resistant animals. What do you plan to do during the next reporting period to accomplish the goals?We will generate the IAV receptor null pigs for testing resistance to IAV We also plan to knockin a interferon responsive MX cassette into the safe harbor locus.
Impacts
What was accomplished under these goals?
Aim-1: Flu decoys were assembled and were introduced into porcine zygotes and modified animals are currently being generated. Aim-2: Genome editors were generated and validated for injections into zygotes to generate ST6- and ST3GAL1 double knockout pigs. The embryo transfers will be attempted in January. Aim-3: Nothing to report here
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Whitelaw CB, Sheets TP, Lillico SG, Telugu BP (2015). Engineering large animal models of human disease. J Pathol. [Epub ahead of print] Review. PubMed PMID: 26414877
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