Fertility testing of preserved epididymal sperm by microinjection: A model for the rescue and utilization of genetically superior animals.

Background: Epididymal sperm preservation is a simple conservation approach that can help prevent the loss of high genetic quality of farm animals. The chance of loss increases, especially during disease outbreaks or other interruptions to normal reproduction function. Aim: This study looked into the ability of preserved ram epididymal sperm to fertilize oocytes. Due to motility becoming an issue following sperm storage for fertilization, the sperm microinjection known as intracytoplasmic sperm injection approach was employed. Methods: The study was divided into two parts. First, involved the preservation of epididymal sperm at 5°C for 12 days. During preservation, sperm quality parameters namely motility, viability, intact membrane, acrosome, and Deoxyribonucleic acid (DNA) are evaluated every three days. For the fertility test in the second experiment, matured oocytes were injected with immotile sperm discovered in the last days of preservation. The presence of pronucleus development following in vitro culture is used as an indicator of sperm's ability to activate and fertilize oocytes. Results: All sperm quality parameters significantly (p < 0.05) declined during preservation time. On day 12, motility was discovered to be 0%, but viable sperm, sperm with intact membrane, acrosome, and DNA remained at 41.86% ± 9.30%, 31.18% ± 5.15%, 21.88% ± 1.93%, and 33.35% ± 8.74%, respectively. On the fertility test, we inject immotile sperm from day 12 of preservation, which has the lowest motility found, into matured oocytes. Those sperms are able to activate (52.05% ± 7.15%) and fertilize (31.37% ± 1.75%) the injected oocytes, but their fertilizing ability is significantly lower (p < 0.05) when compared to the sperm derived from the ejaculate. Conclusion: In this study, simple preservation of epididymal sperm reduces all sperm quality criteria, particularly motility. Using the microinjection approach preserved sperm which had no motility, still demonstrated its ability to activate and fertilize the oocytes. According to that, this study provides potential approaches and tools for using genetically superior animals that have lost their ability to execute regular fertilization, and also prolong reproduction function.

Efficient editing BMP15 in porcine oocytes through microinjection of CRISPR ctRNP.

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/µL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/µL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.

Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination.

Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs.

Comparison of the efficiency and precision of Base editor and CRISPR/Cas9 for inducing defined point mutation (S395F) in ovine embryos.

Cytosine base editors (CBEs) and CRISPR/Cas9-mediated HDR method both have the ability to introduce nucleotide substitution into genomes, which exhibit great potential for improving economically important traits in livestock species. The FecGH mutation (g. C1184T, p. S395F) of growth differentiation factor 9 (GDF9) gene increases prolificacy in Cambridge sheep and Belclare sheep. In the present study, we aimed to compare the efficiency and precision of BE4-Gam and CRISPR/Cas9 systems on generating FecGH mutation in ovine genome. First, the microinjection of BE4-Gam mRNA had no adverse effects on development rate after cleavage, and the efficiencies of total mutants and targeted mutants were 8.9% and 7.1%, respectively. Then, the total mutation and targeted mutation rates were improved from 8.5% to 22.5% (p < .01), and 6.4% to 16.3%, respectively, by adjusting the injection time of BE4-Gam mRNA from 14 to 12 hr post-insemination (hpi). Furthermore, CRISPR/Cas9-mediated HDR method introduced the FecGH mutation at the efficiency of 16.1%, which was comparable to BE4-Gam system (16.3%). There was no bystander editing event happened in edited embryos caused by CRISPR/Cas9, but the bystander editing efficiency was as high as 15.0% in BE4-Gam-edited embryos. In summary, our findings demonstrated that CRISPR/Cas9-mediated HDR method was more accurate than BE4-Gam system in introducing FecGH into ovine genome, and highlight the potential of the former strategy to modify economically important trait-associated SNPs.

Effect of ARTEMIS (DCLRE1C) deficiency and microinjection timing on editing efficiency during somatic cell nuclear transfer and in vitro fertilization using the CRISPR/Cas9 system.

The ability to efficiently introduce site-specific genetic modifications to the mammalian genome has been dramatically improved with the use of the CRISPR/Cas9 system. CRISPR/Cas9 is a powerful tool used to generate genetic modifications by causing double-strand breaks (DSBs) in DNA. Artemis (ART; also known as DCLRE1C), is a nuclear protein and is essential for DSB end joining in DNA repair via the canonical non-homologous end joining (c-NHEJ) pathway. In this work, we tested whether ART deficiency affects DNA repair following CRISPR/Cas9 induced DSBs in somatic cells. We also demonstrated the effect of microinjection timing on embryo developmental ability and gene targeting efficiency of CRISPR/Cas9 system to disrupt the interleukin 2 receptor subunit gamma (IL2RG) locus using porcine in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryos. In comparison to non-injected controls, CRISPR/Cas9 injection of IVF derived zygotes at 4 h and 8 h after fertilization did not impact cleavage and blastocyst rate. Gene modification rate was observed to be higher, 53.3% (9/16) in blastocysts injected 4 h post-fertilization as compared to 11.1% (1/9) in blastocysts injected 8 h post-fertilization. Microinjection 8 h after chemical activation of SCNT derived embryos decreased blastocyst development rate compared to non-injected controls but showed a higher gene modification efficiency of 66.7% as compared to 25% in the 4 h post-activation injection group. Furthermore, we observed that male ART-/- and ART+/- porcine fetal fibroblast (pFF) cells showed lower modification rates (2.5% and 1.9%, respectively) as compared to the ART intact cell line (8.3%). Interestingly, the female ART-/- and ART+/- pFF cells had modification rates (4.2% and 10.1%, respectively) similar to those seen in the ART intact cells. This study demonstrates the complex effect of various parameters such as microinjection timing and ART deficiency on gene editing efficiency in in vitro derived porcine embryos.

Optimizing injection time of GFP plasmid into sheep zygote.

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6-8 hpi, n: 120; 8-10 hpi, n: 122; 10-12 hpi, n: 110 and 12-14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8-10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8-10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.

In vitro development of IVF-derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene.

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus-oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/µl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/µl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/µl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.

A small scale study to evaluate the efficacy of microneedling in the presence or absence of platelet-rich plasma in the treatment of post-clipping alopecia in dogs.

BACKGROUND: Post-clipping alopecia often has a clinically poor response to therapy and prolonged alopecia is a source of anxiety for some owners. In humans and dogs, superficial microtrauma via a microneedling (MN) device induces mechanical stimulation of the hair follicle with resultant hair regrowth. Human studies suggest that concurrent application of platelet-rich plasma (PRP) with MN induces more rapid regrowth of better-quality hair than microneedling alone. HYPOTHESIS: Microneedling with PRP will induce more rapid regrowth of better quality hair. ANIMALS: Four unrelated client-owned dogs diagnosed with post-clipping alopecia. METHODS AND MATERIALS: This was a prospective study. The affected site was divided in half, with the first half treated with MN alone and the second half treated with MN + PRP. Hair regrowth was assessed by clinician and owner using a hair growth assessment scale (HGAS) at one, three, six and 12 months. RESULTS: At three months, all dogs had improved and three exhibited greater hair regrowth on the MN + PRP side. A similar response was noted bilaterally in three dogs, which had improved by 76-100% at six months and remained unchanged at 12 months. One dog improved by < 26% at six months, but had> 50% re-growth by 12 months. The small sample size precluded statistical analysis. CONCLUSIONS AND CLINICAL IMPORTANCE: In dogs with post-clipping alopecia, MN + PRP appeared to induce more rapid hair regrowth than MN; however, overall results were visibly equivalent by six months regardless of method. Both MN and MN + PRP proved successful for treating post-clipping alopecia.

Next Generation Transgenic Rat Model Production.

The next generation of new genetically engineered rat models by microinjection is described. Genome editors such as CRISPR/Cas9 have greatly increased the efficiency with which the rat genome can be modified to generate research models for biomedical research. Pronuclear microinjection of transgene DNA into rat zygotes results in random multicopy transgene integration events that use exogenous promoters to drive expression. Best practices in transgenic animal design indicate the use of precise single copy transgene integration in the genome. This ideal can be achieved by repair of CRISPR/Cas9 chromosome breaks by homology directed repair. The most effective way to achieve this type of transgenic rat model is to deliver genome modification reagents to rat zygotes by pronuclear microinjection. The keys to success in this process are to obtain fertilized eggs (zygotes) from the rat strain of choice, to purify the microinjection reagents, to deliver the reagents to the eggs by pronuclear microinjection, to use the surgical transfer of microinjected eggs to pseudopregnant rats to obtain G0 founder animals that carry the novel genetic modification. Ultimately the success of new rat models is measured by changes in gene expression as in the expression of a new reporter protein such as eGFP, Cre recombinase, or other protein of interest.

Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange.

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes.

Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding α-1,3-galactosyltransferase (GGTA1), which synthesizes the α-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection.

A Simple and Inexpensive Microinjection System for Zebrafish Embryos and Larvae.

Microinjection is a widely used technique to inject defined volumes and concentrations of substances and explore their physiological function in vivo. The technique has been particularly successful with zebrafish embryos; however, the injection equipment can be relatively expensive and therefore available only to well-funded laboratories. In this study, a simple, cheap, easy-to-assemble, and easy-to-use setup with a straightforward, accurate, and efficacious calibration method is introduced. The accuracy of this calibration method was tested by comparing with the results of calibration methods that are currently used in high-cost systems. Injection success with this low-cost system was verified based on the presence of injected dyes in zebrafish embryos, the absence of any significant morphological and behavioral differences between 3,4,-dichloroaniline-treated and untreated embryos, and larval viability.

Gene Targeting in Rabbits: Single-Step Generation of Knock-out Rabbits by Microinjection of CRISPR/Cas9 Plasmids.

The development of genome editing technology has allowed gene disruptions to be achieved in various animal species and has been beneficial to many mammals. Gene disruption using pluripotent stem cells is difficult to achieve in rabbits, but thanks to advances in genome editing technology, a number of gene disruptions have been conducted. This paper describes a simple and easy method for carrying out gene disruptions in rabbits using CRISPR/Cas9 in which the gene to be disrupted is marked, the presence or absence of off-target candidates is checked, and a plasmid allowing simultaneous expression of Cas9 and sgRNA is constructed. Next, the cleaving activity of candidate sequences is investigated, and assessments are carried out to determine whether the target sequences can be cut. Female rabbits subjected to superovulation treatment are mated with male rabbits and fertilized eggs are collected, and then pronuclear injection of plasmid DNA is performed. The next day, the two-cell stage embryos are transplanted into pseudopregnant rabbits, and offspring are born within approximately 29-30 days. The genomic DNA of the offspring is then examined to check what types of genetic modifications have occurred. With the advent of CRISPR/Cas9, the accessibility of gene disruptions in rabbits has improved remarkably. This paper summarizes specifically how to carry out gene disruptions in rabbits.

A chronic in situ coil system adapted for intracerebral stimulation during MRI in rats.

BACKGROUND: We describe the fabrication and performance of a chronic in situ coil system designed to allow focal brain stimulation in rats while acquiring functional MRI data. NEW METHOD: An implantable receive-only surface radiofrequency coil (iCoil) was designed to be fitted subcutaneously, directly onto to the rat skull surface during the intracerebral cannulation procedure. The coil is fixed in place using acrylic dental cement anchored to four screws threaded into the skull. To demonstrate the use of this coil system in situ, whole-brain functional MRI scans were acquired during various stimuli, including intracranial microinfusions of bicuculline and morphine in the prefrontal cortex and ventral tegmental area, respectively. RESULTS/COMPARISON TO OTHER METHODS: SNR performance of the iCoil was superior to three commercially-available coils, in some instances by a factor of two. Widespread BOLD activation was observed in response to bicuculline and morphine microinfusions. CONCLUSION: A new approach was demonstrated for high-SNR MR imaging of the brain in rats with intracranial implants using an implantable surface coil. This approach enables mapping the functional response to highly targeted stimuli such as intracranial microinfusions.

Microstructured Surface Arrays for Injection of Zebrafish Larvae.

Microinjection of zebrafish larvae is an essential technique for delivery of treatments, dyes, microbes, and xenotransplantation into various tissues. Although a number of casts are available to orient embryos at the single-cell stage, no device has been specifically designed to position hatching-stage larvae for microinjection of different tissues. In this study, we present a reusable silicone device consisting of arrayed microstructures, designed to immobilize 2 days postfertilization larvae in lateral, ventral, and dorsal orientations, while providing maximal access to target sites for microinjection. Injection of rhodamine dextran was used to demonstrate the utility of this device for precise microinjection of multiple anatomical targets.

Live-cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods.

The zebrafish model has been used to determine the role of vertebrate innate immunity during bacterial infections. Here, we compare the in vivo immune response induced by GFP-tagged Salmonella Typhimurium inoculated by immersion and microinjection in transgenic zebrafish larvae. Our novel infection protocols in zebrafish allow live-cell imaging of Salmonella colonization.

Skin Vaccination against Rotavirus Using Microneedles: Proof of Concept in Gnotobiotic Piglets.

Live-attenuated oral rotavirus (RV) vaccines have lower efficacy in low income countries, and additionally are associated with a rare but severe adverse event, intussusception. We have been pursuing the development of an inactivated rotavirus vaccine (IRV) using the human rotavirus strain CDC-9 (G1P[8]) through parenteral immunization and previously demonstrated dose sparing and enhanced immunogenicity of intradermal (ID) unadjuvanted IRV using a coated microneedle patch in comparison with intramuscular (IM) administration in mice. The aim of this study was to evaluate the immune response and protection against RV infection and diarrhea conferred by the administration of the ID unadjuvanted IRV using the microneedle device MicronJet600® in neonatal gnotobiotic (Gn) piglets challenged with virulent Wa G1P[8] human RV. Three doses of 5 µg IRV when administered intradermally and 5 µg IRV formulated with aluminum hydroxide [Al(OH)3] when administered intramuscularly induced comparable rotavirus-specific antibody titers of IgA, IgG, IgG avidity index and neutralizing activity in sera of neonatal piglets. Both IRV vaccination regimens protected against RV antigen shedding in stools, and reduced the cumulative diarrhea scores in the piglets. This study demonstrated that the ID and IM administrations of IRV are immunogenic and protective against RV-induced diarrhea in neonatal piglets. Our findings highlight the potential value of an adjuvant sparing effect of the IRV ID delivery route.

Effects of downregulating GLIS1 transcript on preimplantation development and gene expression of bovine embryos.

Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos.

Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development.

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.

Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids.

The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.