Progress 12/15/18 to 12/14/21
Outputs
Target Audience:Target audiences reached include undergraduate students, graduate students, third and fourth year veterinary students and veterinary residents, the two managers of the swine teaching and research facility, and California pork producers. Changes/Problems:A major problem encountered early was renewal of our embryo transfer animal protocol with the insistence by our local IACUC that we prophylactically treat recipients with banamine and bupivacaine despite their known effects as a cyclooxygenase inhibitor and on muscle function, apparently incompatible with the need for normal oviduct transport. After demonstrating an 11% pregnancy rate to day 21 following embryo transfer, IACUC removed the restriction, allowing us to successfully establish pregnancies again. As with all others, the pandemic obviously interfered. Although we cannot yet conclude that nonfunctionality of SRD5A2 is incompatible with fetal development, our lack of success in generating offspring led to evaluation of gene expression in early fetuses and examination of potential off-target edits with none found. What opportunities for training and professional development has the project provided?Two graduate students have developed expertise with in vitro oocyte maturation, in vitro fertilization, electroporation of embryos and evaluation of off-target effects. Two additional graduate students gained some experience with the process. Five undergraduate students developed increased understanding of these processed during their assistance with various aspects of this project. Multiple third and fourth year veterinary students have received training in surgical embryo transfer in pigs; multiple veterinary residents have gained significant experience in surgical embryo transfer. How have the results been disseminated to communities of interest?At this point in time, written communication of results includes an abstract at a national meeting and a dissertation. More informal dissemination includes discussions with undergraduates in a high enrollment undergraduate class in reproductive physiology and dissemination at the California Pork Producers meeting. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Six guides were designed and tested for embryonic survival and efficacy in editing the SRD5A2 gene in zygotes using either zygote injection, electroporation of zygotes, or both. Blastocysts were not obtained following insertion of two of the guides but the remaining four guides were compatible with in vitro development to blastocysts in our experiments. Primers were also designed to produce an approximate 1000 bp product with the site of the edit in approximately the center of the produce in the unedited porcine blastocyst in order to evaluate edits. Editing rate was 64% for the guide introduced via microinjection (and 1 of 2 for the same guide electroporated into blastocysts). The editing rates were 75% to 91% for electroporation of zygotes with a single guide and 100% in blastocysts that developed from 8 zygotes electroporated with a combination of 2 guides targeting the first exon. Pregnancies were established in recipients receiving micrinjected or electroporated embryos; however ultrasound evaluation after day 45 did not detect the presence of fetuses. No offspring were born although a limited amount of placental tissue was recovered from one recipient. When electroporated fetuses were recovered at day 24 of pregnancy, survival of SRD5A2 edited fetuses appeared low relative to other targeted genes with both male and female fetuses represented. Edits were relatively small and all were biallelic. Evaluation of unedited 19 and 26 day fetuses demonstrated that SRD5A2 was expressed in both male and female fetuses at both ages. Hence, expression of SRD5A2 may be important in fetal development. Alternatively, more recent edits of the pig genome indicate two additional genes (one in the positive strand and one in the negative strand) are at least currently mapped to the intronic region of SRD5A2, transcription of which might be affected by editing of SRD5A2. The 12 most likely off-target sites for these guides were identified using three off target software programs. Primers were designed, tested on unedited sequences to verify identity of sequences in these potential off-target sites and subsequently tested on edited blastocysts and fetuses. No off-target edits were identified at any of these 12 sites, which indicates that off-target editing is not a particular issue and that off target edits do not explain the lower survival of edited conceptuses to the fetal stage.
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Progress 12/15/19 to 12/14/20
Outputs
Target Audience:Students gaining practical experience with pigs and advanced veterinary students developing expertise with pigs Changes/Problems:Research was suspended during "shelter in place" orders. The maintenance of the pregnant state despite the loss of fetuses was surprising and has become a topic for additional observation. What opportunities for training and professional development has the project provided?Graduate students and an undergraduate student have developed skill with in vitro maturation and electroporation of pig oocytes. Veterinary residents have gained additional experience with surgical embryo transfer in pigs. 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?Additional embryo transfers will be performed. The plan is to compare efficacy of the three current edits. Maintenance of pregnancy will be evaluated. Assuming that live offspring are produced, deletion of gene expression will be evaluated and the corresponding physiology and effect on boar taint studied in the offspring.
Impacts
What was accomplished under these goals?
A goal of this project is to determine whether elimination of expression of SRD5A2 in intact males will eliminate boar taint and concomitantly, the need to castrate male pigs intended for food consumption. Females that conceived after receiving SRD5A2 deleted, in vivo-produced embryos produced with the first guide studied did not retain fetuses to term but did maintain a pregnancy physiology to term including delivery of small amounts of placenta-like tissue. Three additional guides were designed and demonstrated to be effective in deleting a portion of exon 1 or exon 2 in zygotes that developed to blastocysts. A primer set was designed to evaluate remaining sequence in exon 2. Two pregnancies have been established with embryos receiving this second group of edits. The question remains whether these pregnancies will be maintained to term to produce live offspring, to produce a pregnant state without surviving fetuses, or lost.
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Progress 12/15/18 to 12/14/19
Outputs
Target Audience:Undergraduate students, graduate students, veterinary students, and residents working in laboratory and at UC Davis swine center. Changes/Problems:Our institutional IACUC initially insisted on significant post transfer analgesia, which appeared to interfere with establishment of pregnancy following oviductal transfer. With the elimination of the unneeded and detrimental post-transfer analgesia, initial pregnancy rate has soared. What opportunities for training and professional development has the project provided?Undergraduate and graduate students in our laboratory and veterinary students and residents have become aware of the project, the need for the project, and the approach to accomplish the objectives. How have the results been disseminated to communities of interest?Other than a presentation at the AAVMC Gene Editing summit and informal conversations with students, there is currently nothing to report. What do you plan to do during the next reporting period to accomplish the goals?We are actively working on embryo transfer of in vitro matured, in vitro fertilized, electroporated embryos to generate pregnancies that will reach term in order to generate the pigs for the remaining objectives.
Impacts
What was accomplished under these goals?
We have evaluated the efficiency of gene editing by three guide RNAs and selected one of those guides to assist us in completing the remaining objectives.
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