Progress 07/01/23 to 06/30/24
Outputs
Target Audience:Academic and industry scientists working on animal health, especially with respect to genetics and disease transmission. Changes/Problems:We unfortunatley were unsuccessful in transfecting the two yeast assemblon vector DNAs produced by the Boeke lab into chicken cells to produce infectious virus. The Boeke lab is working on a full-length vector and the Conrad lab has circumvented this obstacle by utilizing the red-mediated recombination method to delete the target genes we identified in Objective 1. What opportunities for training and professional development has the project provided?Thus far this project has provided training for a postdoc and Master's student on MDV YAC engineering attempts. We currently have one postdoc FTE whose main objective is to work on objectives 2 and 3 of this project. How have the results been disseminated to communities of interest?So far, a summary of our results has been presented the last 3 years at the Conference of Research Workers in Animal Diseases (CRWAD)in Chicago, January of eachyear. A detailed version of the MDV knockout production was presented this past June at the American Society for Virology Conference 2024 in Columbus (OH). Our latest results using lentiviruses to deliver and express target MDV genes in avian macrophage-like cells will be presented at the International Marek's Disease and Avian Herpesvirus Conference 2024 in St Louis (MO). What do you plan to do during the next reporting period to accomplish the goals?Objective 1. The first objective is complete as our screening system found seven MDV genes with IFN-I immunomodulatory capacity. We are working on including our results in a scientific publication. Objective 2. Now that MDV target genes have been identified, the Conrad laboratory will continue their efforts on developing an assay based on CRISPR/Cas9 to identify and validate the functional domain on each gene that inhibits the production of type I IFNs. Objective 3. We are hoping to determine the virulence of our double deletion mutants during the fall of 2024 and include the results (together with the results from objective 1) in a scientific publication. If the virulence assays are satisfactory, we will test the MD vaccinal protective efficacy of the knockouts during spring 2025.
Impacts
What was accomplished under these goals?
Objective 1: Identify the MDV virulence factors that suppress the production of type I IFNs during viral infection (Dunn, Conrad and Boeke labs). In a collaborative effort, the Conrad and Dunn laboratories developed and optimized an assay for identification of MDV immunomodulatory genes based on the activation of avian macrophages expressing target MDV genes via lentivirus integration. We purchased five different lentiviruses carrying genes US3, UL46, UL48, Meq, R-LORF-4 and a control GFP gene (under control of a mammalian expression promotor) from Vector Builder. Additionally, each lentivirus also carried a puromycin resistance cassette that makes the cells resistant to puromycin upon lentivirus integration (as a selection aid). We infected chicken macrophage-like HD11 cells with each of the lentivirus strains (MOI 10) following Vector Builder specifications. We exposed infected HD11 to 4ug/ml of puromycin for two weeks (replacing with drug-containing media every three days). Cells that showed resistanceto puromycin were expected to have a lentivirus integrated in their genome and therefore expression of inserted MDV gene. Lentivirus integration is random andis expected to occur at different sites within the avian genome in each cell. For that reason, after lentivirus insertion we performed a clonal selection in which we plated two 96-well plates with one single cell per well and we left the cells replicate and start a clonal population for a period of 10 days (replacing with drug-containing media every three days, 3.2 μg/mL puromycin). After that time, we selected five wells that showed only one clonal resistant population and kept expanding them. We have developed a system in which we promote the production of IFN in HD11 cells by transfecting them with a 3μg of a 2kb double-stranded DNA fragment. We used qPCR to determine the gene expression levels of the IFNω1 gene and we identified that the expression of the five MDV genes produce a significant decrease in the expression of IFNω1 compared to the control GFP. The Boeke lab (subaward) obtained the MDV containing BAC rMd5B40 from the prime awardee, subsequent to testing it for infectivity, and resequencing the BAC.To enable the editing of the BAC rMd5B40, containing the ~175 kb Md5 strain MDV, we focused on designing a version that is able to replicate in yeast, thereby enabling HR mediated cloning in yeast. The entire MDV BAC DNA was subcloned into their lab's go-to vector for large DNA - performing a backbone swap from the current BAC backbone to our pLM1050 backbone. The Boeke lab now has the entire genome cloned on two yeast assemblon vectors. Using this vector, we have knocked out 5 genes of interest with URA3.Transfection of the DNAs into chicken cells to confirm infectious virus was unsuccessful. Objective 2: Identify and validate the functional domain of each virulence factor that inhibits the production of type I IFNs. (Conrad lab). We will be able to start work on this objective once we have a transformed avian monocytic cell line in which the IFNB gene is replaced by a fluorescent reporter but still driven by the native IFNB control elements by CRISPR/Cas9 deletion and insertion. Our first attempt to create this line failed to produce clones. We are currently producing more clones (as described above). Objective 3: Test defined recombinant MDVs for virulence and MD vaccinal protective efficacy (Dunn and Conrad labs). As the use of the YAC/BAC system developed in Dr. Boeke's lab did not result in successful viral recovery, the Conrad lab decided to change our mutant production strategy and used the Tischer et al. two-step Red-mediated recombination method to delete some of the target genes identified in objective 1. We specifically used the Md5B40BAC?meq produced by Dr Dunn and collaborators in 2010 and subsequently modified by T. Kim who deleted the TRL-TRS region (unpublished). This BAC contains the complete Md5 genome with a deletion of the meq gene and the TRL-TRS region containing one of the two long inverted repeat regions of the MDV genome. With this strategy we successfully produced and rescued three MDV double mutants: (i) Md5-B40?meq?UL46, Md5-B40?meq?UL48, and Md5-B40?meq?R-LORF4. The Dunn labis preparing to test the virulence of the knockout mutants in chickens.
Publications
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:Academic and industry scientists working on animal health, especially with respect to genetics and disease transmission. Changes/Problems:The COVID-19 pandemiccaused delays in meeting planned objectives due to significant obstacles in hiring support staff and acquiring critical reagents due to supply chain shortages.Now that we have the virus cloned into yeast this will allow rapid progress in generating and evaluating our recombinant viruses for virulence factors that inhibit production of type I IFN. What opportunities for training and professional development has the project provided?Thus far this project has provided training for a postdoc and Master's student on MDV YAC engineering attempts. We currently have one postdoc FTE whose main objective is work on objectives 2 and 3 of this project. How have the results been disseminated to communities of interest?Results have been disseminated at the Conference of Research Workers in Animal Diseases, Chicago, IL. Project is ongoing. What do you plan to do during the next reporting period to accomplish the goals?Continue as planned and then write report results in scientific publications. The Dunn and Conrad labs are continuing efforts to produce clones expressing the relevant (see above) MDV immunomodulatory genes and we expect to test those as soon as clonal selection is finished.
Impacts
What was accomplished under these goals?
Objective 1: Identify the MDV virulence factors that suppress the production of type I IFNs during viral infection (Dunn, Conrad and Boeke labs). The Dunn laboratory has verified the infectivity of the MDV rMd5B40 BAC clone and is attempting to produce lentivirus-transfected clones which express the relevant MDV immunomodulatory genes. Thus far we have transfected HD11 (avian monocyte-like cells) with lentivirus constructs expressing the following MDV genes: US3, UL18, UL41, UL46 and UL48. We are currently in the process of selecting these clones. The Boeke lab (subaward) obtained the MDV containing BAC rMd5B40 from the prime awardee, subsequent to testing it for infectivity, and resequencing the BAC.To enable the editing of the BAC rMd5B40, containing the ~175 kb Md5 strain MDV, we focused on designing a version that is able to replicate in yeast, thereby enabling HR mediated cloning in yeast. The entire MDV BAC DNA was subcloned into their lab's go-to vector for large DNA - performing a backbone swap from the current BAC backbone to our pLM1050 backbone. The Boeke lab now has the entire genome cloned on two yeast assemblon vectors. Transfection of the DNAs into chicken cells to confirm infectious virus is underway. Using this vector, we have knocked out 5 genes of interest with URA3. The Conrad laboratory is working on two parallel projects related to this objective. First, we are trying to reconstitute the MDV deletion mutants produced by Dr. Boeke's lab. We received seven BACs that we will use for the reconstitution of five MDV knockout mutants lacking genes US3, UL18, UL41, UL46 and UL48. We already started trying to reconstitute mutant US3. For this mutant, the MDV genome was cloned into two different BACs. We sent the BACs for long-read next generation sequencing to verify that all the genomic pieces needed for MDV reconstitution were present (results not yet received). So far we have tested two different protocols to introduce the BACs into duck embryonic fibroblasts (DEFs). In approach 1 we used the transfection reagent Turbofect 8.0 and the BACs were not linearized. In approach 2 we linearized the BACs using endonuclease NotI (single cutter enzyme) and we tried to introduce them in DEFs by electroporation. Neither approach worked, and we believe the failure is due to the low BAC concentration used in the experiments. We are currently working on getting higher concentration BAC extracts to repeat both strategies to determine which one is more efficient. For the second project connected to this objective, we are working on expressing the same genes that were deleted by Dr. Boeke's laboratory in the chicken macrophage-like cell line HD11 to determine the extent of their IFN-related immunomodulation role. We purchased seven different lentiviruses carrying genes US3, US9, UL18, UL41, UL46, UL48 and a control GFP gene (under control of a mammalian expression promotor) from Vector Builder. Additionally, each lentivirus also carried a puromycin resistance cassette that makes the cells resistant to puromycin upon lentivirus integration (as a selection aid). We infected HD11 with each of the lentivirus strains (MOI 10) and following Vector Builder specifications. We exposed infected HD11 to 4ug/ml of puromycin for two weeks (replacing with drug-containing media every three days). Cells showed resistant to puromycin were expected to have a lentivirus integrated in their genome. Lentivirus integration is random it is expected to occur at different sites within the avian genome in each cell. For that reason, after lentivirus insertion we performed a clonal selection in which we plated two 48-well plates with one single cell per well and we left the cells replicate and start a clonal population for a period of 10 days (replacing with drug-containing media every three days, 3.2 μg/mL puromycin). After that time, we selected five wells that show only one clonal resistant population and kept expanding them. We have developed a system in which we promote the production of IFN inHD11 cells by transfecting them with a 3μg of a 2kb double-stranded DNA fragment. In the coming weeks we are going to use this system to test the different MDV-gene-expressing HD11 and see if the expression of each of the MDV genes impact the production of the interferon compared to the GFP-expressing HD11 control. Objective 2: Identify and validate the functional domain of each virulence factor that inhibits the production of type I IFNs. (Conrad lab). We will be able to start work on this objective once we have a transformed avian monocytic cell line in which the IFNB gene is replaced by a fluorescent reporter but still driven by the native IFNB control elements by CRISPR/Cas9 deletion and insertion. Our first attempt to create this line failed to produce clones. We are currently producing more clones (as described above). Objective 3: Test defined recombinant MDVs for virulence and MD vaccinal protective efficacy (Dunn and Conrad labs). This objective involves the use of synthesized BACs in live animals and has not been started yet.
Publications
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:Academic and industry scientists working on animal health, especially with respect to genetics and disease transmission. Changes/Problems:The COVID-19 pandemic has caused delays in meeting planned objectives due to significant obstacles in hiring support staff and acquiring critical reagents due to supply chain shortages. What opportunities for training and professional development has the project provided?Thus far this project has provided training for a postdoctoral research associateand Master's student on MDV YAC engineering attempts. We have selected another postdoctoral research associate whose main objective will be work on objectives 2 and 3 of this project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Continue as planned and then write report results in scientific publication. The Boeke lab is attempting to sub clone the entire MDV BAC DNA into their lab's go-to vector for large DNA - performing a backbone swap from the current BAC backbone to our pLM1050 backbone. The Dunn lab is continuing efforts to produce a transformed avian monocytic cell line in which the IFNB gene is replaced by a fluorescent reporter but still driven by the native IFNB control elements by CRISPR/Cas9 deletion and insertion.
Impacts
What was accomplished under these goals?
Objective 1: Identify the MDV virulence factors that suppress the production of type I IFNs during viral infection (20% completion). The Dunn laboratory has verified the infectivity of the MDV rMd5B40 BAC clone and attempted to produce a transformed avian monocytic cell line in which the IFNB gene is replaced by a fluorescent reporter but still driven by the native IFNB control elements by CRISPR/Cas9 deletion and insertion, which failed to produce clones. We are currently attempting to do this again. The Boeke lab (subaward) obtained the MDV containing BAC rMd5B40 from the prime awardee, subsequent to testing it for infectivity, and resequencing the BAC. To enable the editing of the BAC rMd5B40, containing the ~175 kb Md5 strain MDV, we have focused on designing a version that is able to replicate in yeast, thereby enabling HR mediated cloning in yeast. We have been joined on the project by a Master's student Maria Bahamon from Colombia, who is a Master's student in the Biotechnology program at the Tandon School of Engineering. Ms. Bahamon has learned a great deal about working with yeast and has constructed a number of highly useful big DNA constructs. Ms. Bahamon, mentored by Max Haase, has been making multiple attempts to engineer the MDV vector (a BAC ) for manipulation in yeast. The following paragraphs describe the work done over the past year by Ms. Bahamon for MDV YAC engineering attempts. Insertion of CEN/ARS fragment and LEU2 fragment from pRS415 into the BAC. First, BY4742 was transformed with Cas9 expressing vector. Next this strain with Cas9 was transformed with the MDV BAC DNA, a gRNA plasmid with guide targeting the MDV BAC, and an amplicon consisting of ARS/CEN-LEU2 with 25 bp of homology to the MDV BAC. The first attempt of yeast transformation yielded no colonies. We freshly prepared additional MDV BAC with the NucleoBond Xtra BAC kit and redid the transformation. The second attempt at the transformation yielded colonies but the PCR genotyping showed absence of the BAC DNA. Colonies we likely circularization products of the CEN/ARS-Leu2 fragment. With the aim to avoid the CEN/ARA-Leu2 mini circle, we constructed a new plasmid to cut at two different parts of the BAC, with homology directed insertion of the CEN/ARS at site one and the LEU2 fragment at site two. The yeast transformation was attempted as before, but it again yielded no colonies. It was tried again with a higher concentration of the BAC, which yielded a single colony. This single colony initially scored as a positive clone by PCR genotyping. However, upon restreaking the clone, we observed a consistent loss of the BAC DNA. The transformation was tried again with even more BAC DNA (1ug, 2ug), which yielded more colonies, ten colonies with presence of the BAC DNA were re streaked and sequenced. However, as before all restreaked clones consistently lost the MDV BAC DNA and all sequenced clones showed total lack of MDV BAC DNA. Currently the Boeke lab is attempting to sub clone the entire MDV BAC DNA into their lab's go-to vector for large DNA - performing a backbone swap from the current BAC backbone to our pLM1050 backbone. These experiments are currently underway. Objective 2: Identify and validate the functional domain of each virulence factor that inhibits the production of type I IFNs. (0% completion). We will be able to start work on this objective once we have a transformed avian monocytic cell line in which the IFNB gene is replaced by a fluorescent reporter but still driven by the native IFNB control elements by CRISPR/Cas9 deletion and insertion. Our first attempt to create this line failed to produce clones. As a substitute for the described construct will produce a stably-expressing cell line in HD11 cells which will contain a nano-luciferase reporter driven by the native IFNB control elements but which as the entire cassette randomly inserted into the HD11 cell genome. This cell line will be used to test the infectious MDV rMd5B40 constructs produced by the Boeke laboratory in Objective 1. Objective 3: Test defined recombinant MDVs for virulence and MD vaccinal protectiveefficacy (Dunn and Boeke labs). This objective involves the use of synthesized BACs in live animals and is pending products from objectives 1 and 2.
Publications
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Progress 07/01/20 to 06/30/21
Outputs
Target Audience:Academic and industry scientists working on animal health, especially with respect to genetics and disease transmission. Changes/Problems:The most significant obstacle has been our agency policy on number of people and hours allowed in the laboratory due to COVID-19. This has also led to a delay in hiring of our postdoc researcher. We are hopeful that these restrictions will soon be lifted and we can increase our efforts and catch up for lost time in the laboratory. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Continue as planned and then write report results in scientific publication.
Impacts
What was accomplished under these goals?
Objective 1: Identify the MDV virulence factors that suppress the production of type I IFNs during viral infection (10% completion). The Dunn laboratory has verified the infectivity of the MDV rMd5B40 BAC clone and supplied it to the Boeke laboratory (subawardee). We have previously identified a list of 10 promising genes which have been reported to inhibit the production of IFN-Is, but thus far we have not been able to access laboratory time to test them. The Boeke lab obtained the MDV containing BAC rMd5B40 from the Dunn laboratory subsequent to testing it for infectivity and resequencing the BAC. To enable the editing of the BAC rMd5B40, containing the ~175 kb Md5 strain MDV, we have focused on designing a version that is able to replicate in yeast, thereby enabling HR mediated cloning in yeast. Our initial approach to establish a replicating BAC in yeast has met with some difficulties. Our approach was to target the terminal region near the loxP site on the pBeloBAC11 backbone with a gRNA to linearize the BAC. This CRISPR mediated cut was done as part of a co-transformation in yeast, with a linear DNA fragment encoding the selectable marker/origin LEU2/ARS-CEN bearing appropriate terminal homologies to the linearized BAC. Transformation followed by selection on medium lacking leucine led to the isolation of ~100 colonies. PCR screening for novel BAC-Leu2 junctions highlighted 10 clones that had potentially assembled the BAC-yeast DNA fragments. We attempted to verify all 10 by WGS to ensure the entire infectious BAC insert remained. Unfortunately, all sequenced clones lacked reads mapping to the BAC sequence of interest, thus the clones seemingly lost the infectious BAC DNA. We are now attempting to construct the base BAC clone using alternative strategies. With this in hand, we have developed a strategy to further engineer the virus genome in this construct using CRISPR in yeast, to replace the viral meq sequences. Simply knocking out both meq sequences from MDV will result in a protective virus from a vaccine perspective. Specifically, we plan to insert a CMV promoter-driven FusionRed in one of the meq sites, and a CMV-driven nano-luciferase in the OTHER meq site. This would make the virus every easy to see and track both in vitro and in vivo (because of mCherry) and also very easy to quantitate total replication in culture because of the nano-luc insert. At each stage of engineering the virus containing BAC will be sent recovered to bacteria and shipped back to the prime contractor to evaluate infectivity and other characteristics of the virus (e.g., replication kinetics). Objective 2: Identify and validate the functional domain of each virulence factor that inhibits the production of type I IFNs. (0% completion). We will be able to start work on this objective once we have access to the laboratory, and once we have a transformed avian monocytic cell line in which the IFNB gene is replaced by a fluorescent reporter but still driven by the native IFNB control elements by CRISPR/Cas9 deletion and insertion. Our first attempt to create this line failed to produce clones. As a substitute for the described construct we will produce a stably-expressing cell line in HD11 cells which will contain a nano-luciferase reporter driven by the native IFNB control elements but which as the entire cassette randomly inserted into the HD11 cell genome. This cell line will be used to test the infectious MDV rMd5B40 constructs produced by the Boeke laboratory. Objective 3: Test defined recombinant MDVs for virulence and MD vaccinal protective efficacy (Dunn and Boeke labs). This objective involves the use of synthesized BACs in live animals and has not been started yet.
Publications
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