Transgenic Founder Research Articles

Differences in Susceptibility to SARS-CoV-2 Infection Among Transgenic hACE2-Hamster Founder Lines.

Animal models that are susceptible to SARS-CoV-2 infection and develop clinical signs like human COVID-19 are desired to understand viral pathogenesis and develop effective medical countermeasures. The golden Syrian hamster is important for the study of SARS-CoV-2 since hamsters are naturally susceptible to SARS-CoV-2. However, infected hamsters show only limited clinical disease and resolve infection quickly. In this study, we describe development of human angiotensin-converting enzyme 2 (hACE2) transgenic hamsters as a model for COVID-19. During development of the model for SARS-CoV-2, we observed that different hACE2 transgenic hamster founder lines varied in their susceptibility to SARS-CoV-2 lethal infection. The highly susceptible hACE2 founder lines F0F35 and F0M41 rapidly progress to severe infection and death within 6 days post-infection (p.i.). Clinical signs included lethargy, weight loss, dyspnea, and mortality. Lethality was observed in a viral dose-dependent manner with a lethal dose as low as 1 × 100.15 CCID50. In addition, virus shedding from highly susceptible lines was detected in oropharyngeal swabs on days 2-5 p.i., and virus titers were observed at 105.5-6.5 CCID50 in lung and brain tissue by day 4 p.i.. Histopathology revealed that infected hACE2-hamsters developed rhinitis, tracheitis, bronchointerstitial pneumonia, and encephalitis. Mortality in highly susceptible hACE2-hamsters can be attributed to neurologic disease with contributions from the accompanying respiratory disease. In contrast, virus challenge of animals from less susceptible founder lines, F0M44 and F0M51, resulted in only 0-20% mortality. To demonstrate utility of this SARS-CoV-2 infection model, we determined the protective effect of the TLR3 agonist polyinosinic-polycytidylic acid (Poly (I:C)). Prophylactic treatment with Poly (I:C) significantly improved survival in highly susceptible hACE2-hamsters. In summary, our studies demonstrate that hACE2 transgenic hamsters differ in their susceptibility to SARS-CoV-2 infection, based on the transgenic hamster founder line, and that prophylactic treatment with Poly (I:C) was protective in this COVID-19 model of highly susceptible hACE2-hamsters.

Efficient generation of epigenetic disease model mice by epigenome editing using the piggyBac transposon system

BackgroundEpigenome-edited animal models enable direct demonstration of disease causing epigenetic mutations. Transgenic (TG) mice stably expressing epigenome-editing factors exhibit dramatic and stable changes in target epigenome modifications. Successful germline transmission of a transgene from founder mice to offspring will yield a sufficient number of epigenome-edited mice for phenotypic analysis; however, if the epigenetic mutation has a detrimental phenotypic effect, it can become difficult to obtain the next generation of animals. In this case, the phenotype of founder mice must be analyzed directly. Unfortunately, current TG mouse production efficiency (TG founders per pups born) is relatively low, and improvements would increase the versatility of this technology.ResultsIn the current study, we describe an approach to generate epigenome-edited TG mice using a combination of both the dCas9–SunTag and piggyBac (PB) transposon systems. Using this system, we successfully generated mice with demethylation of the differential methylated region of the H19 gene (H19-DMR), as a model for Silver–Russell syndrome (SRS). SRS is a disorder leading to growth retardation, resulting from low insulin-like growth factor 2 (IGF2) gene expression, often caused by epimutations at the H19-IGF2 locus. Under optimized conditions, the efficiency of TG mice production using the PB system was approximately threefold higher than that using the conventional method. TG mice generated by this system showed demethylation of the targeted DNA region and associated changes in gene expression. In addition, these mice exhibited some features of SRS, including intrauterine and postnatal growth retardation, due to demethylation of H19-DMR.ConclusionsThe dCas9–SunTag and PB systems serve as a simple and reliable platform for conducting direct experiments using epigenome-edited founder mice.

86 Creating homozygous offspring using oviductal sperm deposition with poor quality cryopreserved semen from a transgenic founder goat.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

25th ANNIVERSARY OF CLONING BY SOMATIC CELL NUCLEAR TRANSFER: Generation of genetically engineered livestock using somatic cell nuclear transfer.

Genetic engineering (GE) of livestock initially has been accomplished primarily using pronuclear microinjection into zygotes (1985-1996). The applications of the technology were limited due to low integration efficiency, aberrant transgene expression resulting from random integration and the presence of genetic mosaicism in transgenic founder animals. Despite enormous efforts to established embryonic stem cells (ESCs) for domestic species, the ESC GE technology does not exist for livestock. Development of somatic cell nuclear transfer (SCNT) has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals. For nearly two decades since the birth of Dolly (1996-2013), SCNT was the only method used for the generation of knockout and knockin livestock. Arrival of CRISPRS/Cas9 system, a new generation of gene-editing technology, gave us an ability to introduce precise genome modifications easily and efficiently. This technological advancement accelerated production of GE livestock by SCNT and reinstated zygote micromanipulation as an important GE approach. The primary advantage of the SCNT technology is the ability to confirm in vitro that the desired genetic modification is present in the somatic cells prior to animal production. The edited cells could also be tested for potential off-target mutations. Additionally, this method eliminates the risk of genetic mosaicism frequently observed following zygote micromanipulation. Despite its low efficiency, SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field.

Generation and expression analysis of BAC humanized mice carrying HLA-DP401 haplotype.

BackgroundHuman leukocyte antigen (HLA)‐DP is much less studied than other HLA class II antigens, that is, HLA‐DR and HLA‐DQ, etc. However, the accumulating data have suggested the important roles of DP‐restricted responses in the context of cancer, allergy, and infectious disease. Lack of animal models expressing these genes as authentic cis‐haplotypes blocks our understanding for the role of HLA‐DP haplotypes in immunity.MethodsTo explore the potential cis‐acting control elements involved in the transcriptional regulation of the HLA‐DPA1/DPB1 gene, we performed the expression analysis using bacterial artificial chromosome (BAC)‐based transgenic humanized mice in the C57BL/6 background, which carried the entire HLA‐DP401 gene locus. We further developed a mouse model of Staphylococcus aureus pneumonia in HLA‐DP401 humanized transgenic mice, and performed the analysis on the expression pattern of HLA‐DP401 and immunological responses in the model.ResultsIn this study, we screened and identified a BAC clone spanning the entire HLA‐DP gene locus. DNA from this clone was analyzed for integrity by pulsed‐field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA‐DPA1/DPB1 gene. Transgene copy numbers were determined by real‐time PCR analysis. HLA‐DP401 gene expression was analyzed at the mRNA and protein level. Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA‐DP401‐H2‐Aβ1 humanized mice. Increased expression of HLA‐DP401 was observed in the thymus of the humanized mice infected by S aureus.ConclusionsWe generated several BAC transgenic mice, and analyzed the expression of HLA‐DPA1/DPB1 in those mice. A model of S aureus‐induced pneumonia in the HLA‐DP401‐H2‐Aβ1−/− humanized mice was further developed, and S aureus infection upregulated the HLA‐DP401 expression in thymus of those humanized mice. These findings demonstrate the potential of those HLA‐DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC‐associated immunological diseases.

Generation and Breeding of EGFP-Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling.

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

Cloning of embryos

Recent research clearly shows that fusion genes can be microinjected into a pronucleus of an ovum and integrate into the pig genome. Animals with such fusion genes are called 'transgenic'. The percentage of injected ova that developed into transgenic pigs varied among experiments from 0.31% to 1.73%. The percentage of transgenic pigs that expressed the fusion gene ranged from 17% to 100%. Eleven different regulatory sequences have been used for fusion genes transferred into pigs. Some of these regulatory sequences directed strong gene expression, but control over level of expression was inadequate. Other regulatory sequences directed weak expression, but imparted only brief spikes of induced expression. The predominant gene coding sequences transferred were for growth-related hormones. Elevation of growth hormone (GH) in expressing transgenic pigs enhanced plasma concentrations of insulin-like growth factor-I (IGF-I), insulin, and glucose, improved feed efficiency about 15%, and markedly reduced subcutaneous fat compared to nontransgenic siblings. Growth rate was enhanced in some transgenic GH pigs but not in others, possibly due to dietary limits. The 'over-expression' of GH was detrimental to the general health of most transgenic pigs. The most prevalent problems were lethargy, lameness, and gastric ulcers. Gilts that expressed foreign GH genes were anoestrous. Boars that expressed foreign GH genes lacked libido, but their semen was fertile when used by artificial insemination. Six different fusion genes have been transmitted from transgenic founders to progeny. Most of the transgenic pigs that produced progeny transmitted the fusion gene as an autosomal dominant trait to about half of their progeny. Regulatory sequences that will permit full control of gene expression must be developed before the full potential of gene transfer in pigs can be realized.

154 A method of oviductal semen deposition for use in the goat

The objective of this study was to investigate a method of oviducal semen deposition as a strategy for producing offspring from poor-quality cryopreserved goat sperm. Invitro fertilisation (IVF) and AI are common assisted reproductive technologies used in small ruminants, but they have varied results in the goat. The use of poor-quality cryopreserved-thawed sperm (<50% live/dead ratio at post-thaw) can decrease the rate of success. These procedures were performed in the month of November in Central Massachusetts in the United States (42° N). Seven 10-year-old dairy goats (Saanen, Toggenburg, and Alpine breeds) were synchronised and superovulated using a progesterone implant on Day 0, a prostaglandin injection at Day 7, two daily injections of 36mg of FSH ~12h apart on Days 12-15, and progesterone implant removal on Day 14 followed by an injection of 50µg of gonadotrophin-releasing hormone. Sperm deposition was performed on Day 17 (72 h after implant removal). The animals were anaesthetised using a standardised protocol, intubated, and maintained using isoflurane, and sterile prep was performed before a midline laparotomy procedure. Straws from a single ejaculate from a transgenic founder that was cryopreserved using a commercial two-step glycerol-egg yolk-based extender were used. A straw from this collection was post-thawed 30 days after collection and, using a commercial live/dead stain, 67% live sperm was determined. The optimal type of sperm prep and sperm concentration is unknown and may be dependent on sperm quality. Therefore, different gradient preps using Vitrolife SpermGrad at three volumes (1.5 (used on two animals), 1.0, and 0.5mL) as well as two volumes of IVF Bioscience Bovine BO-SemenPrep (4.0mL (used on two animals) and 2.0mL) were used. All five pellets were diluted in 1.0mL of IVF Bioscience Bovine BO-IVF media. Sperm concentrations ranging from 75×106 to 27×106 spermmL−1 were deposited into one oviduct; then, a 10:1 dilution was performed and 7.5×106 to 2.7×10 spermmL−1 were deposited into the contralateral oviduct. The depositions were performed just proximal to the uterotubal junction in a volume of 0.1mL of diluent via a tuberculin syringe attached to a 20-gauge needle. Two days following the procedure, oviducts were flushed postmortem from three of the seven randomly selected goats. All three had fertilised embryos, and nineteen 8-cell embryos were retrieved. Three of these embryos were surgically transferred to the distal uterine horn of a suitable recipient. The recipient became pregnant and produced a single offspring. The remaining four of seven goats were killed 41 days post-surgery. Two of the four goats were pregnant, with one carrying one fetus and the other carrying five fetuses. Further studies are needed to optimise this method, but these initial results indicate that oviducal semen deposition directly into the oviduct proximal to the uterotubal junction may be a suitable alternative for producing offspring from suboptimal cryopreserved-thawed goat sperm.

Retroelement Insertion in a CRISPR/Cas9 Editing Site in the Early Embryo Intensifies Genetic Mosaicism.

Continued CRISPR/Cas9-mediated editing activity that allows differential and asynchronous modification of alleles in successive cell generations expands allelic complexity. To understand the earliest events during CRISPR/Cas9 editing and the allelic selection among the progeny of subsequent cell divisions, we inspected in detail the genotypes of 4- and 8-cell embryos and embryonic stem cells (ESCs) after microinjection of a CRISPR toolkit into the zygotes. We found a higher editing frequency in 8-cell embryos than in 4-cell embryos, indicating that the CRISPR/Cas9 activity persisted through the 8-cell stage. Analysis of a CRISPR/Cas9 transgenic founder mouse revealed that four different alleles were present in its organs in different combinations and that its germline included three different mutant alleles, as shown by the genotypes of the pups. The indel depth, which measured the extent of indels at the sequence level within single embryos, decreased significantly as the embryos advanced to form ESCs, suggesting that exclusion of fatal indels occurred in the subsequent cell generations. Interestingly, we discovered that the CRISPR sites frequently contained introduced retroelement sequences and that this occurred preferentially with certain classes of retroelements. Therefore, in addition to CRISPR/Cas9's innate mechanism of separate, differential enzymatic modifications of alleles, the frequent retroelement insertions that occur in early mouse embryos during CRISPR/Cas9 editing further expand the allelic diversity and mosaicism in the resulting transgenic founders.

Effects of TLR4 overexpression on sperm quality, seminal plasma biomarkers, sperm DNA methylation and pregnancy rate in sheep

Genetic modification provides a means to enhancing disease resistance in animals. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. To compare semen characteristics including sperm quality, seminal plasma biochemical index, sperm DNA methylation and pregnancy rate of three-year old transgenic sheep with TLR4 overexpressed (toll like receptor 4, TLR4) and non-transgenic ram. Sixteen transgenic ram of F0 generation were produced by microinjection of the TLR4 plasmid into the pronucleus of fertilized ova. Seven transgenic sheep of F1 generation was produced by breeding F0 transgenic founders with non-transgenic sheep of the same breed. There were no significant differences between transgenic and control rams for all semen quality parameters, including semen volume, sperm concentration, sperm viability, and percentages of sperm with an intact plasma membrane, acrosomal integrity, and viable sperm with high mitochondrial membrane potential in both F0 and F1 generation. Furthermore, no significant differences were found for seminal plasma concentrations of zinc, neutral alpha-glucosidase, acid phosphatase or fructose, nor for levels of H19 and IGF2R methylation in sperm DNA. In addition, pregnancy rate was also similar between these two groups. In conclusion, there was no evidence that TLR4 overexpression altered the sperm quality, seminal plasma or sperm DNA of transgenic sheep.

Breeding Strategies for Genetically Modified Mice.

Genetically modified mice can be generated using a variety of methods. Depending on the method used, the breeding strategy must be modified not only for the initial founding generation of mouse, but to establish individual mouse lines as well. Transgenic founder mice are each unique and must be outcrossed to initially establish the line. Mice that have been targeted using embryonic stem cells will need to be tested for germ line transmission of the modified gene prior to establishing a line. Mice that have been targeted using CRISPR/Cas9 gene-editing endonucleases will often be mosaic, and thus, also require testing for germ line transmission of the mutation of interest.

A plan to diversify a transgenic blight‐tolerant American chestnut population using citizen science

Societal Impact StatementOver four billion American chestnut trees have been killed as a result of an introduced pathogen, the chestnut blight fungus. Recently, transgenic blight‐tolerant American chestnut trees have been produced by inserting a gene from wheat into the American chestnut genome. Pending federal approval to use these transgenic trees for large‐scale forest restoration, this would be the first instance where a transgenic approach has been used to restore a tree species that has been rendered functionally extinct by an introduced pathogen. With the help of citizen scientists, we estimate that large‐scale forest restoration using blight‐tolerant American chestnut trees is possible within the next few decades.SummaryBreeding transgenic blight‐tolerant American chestnuts with susceptible wild‐type (WT) trees is potentially an efficient method to rescue the genetic diversity and adaptive capacity of the American chestnut population for large‐scale restoration.To develop a breeding plan to diversify a transgenic blight‐tolerant population, we simulated pedigrees to estimate inbreeding coefficients and effective population size in scenarios involving outcrossing 1–4 transgenic founders to a maximum of 1,500 WT trees over 1–5 generations. We also simulated marker‐assisted introgression scenarios to minimize the extent of the transgenic founder genome, especially on the transgene carrier chromosome.Simulations suggest that the effective population size may be increased to >500, and the average inbreeding coefficient reduced to <0.01, by outcrossing a single transgenic founder over five generations to 2, 25, 50, 150, and 450 (677 total) WT parents. Three generations of marker‐assisted introgression is predicted to decrease the length of the founder genome to between 7% and 13% of the transgene carrier chromosome length as compared to 42% with event selection (ES) only. Transgenic outcross selections may be intercrossed to select progeny homozygous for the transgene for planting in seed orchards.A diversified population of transgenic blight‐tolerant American chestnut is estimated to be available for use in large‐scale forest restoration 20–35 years after federal approval to distribute the trees. In contrast, trees from earlier generations would be available almost immediately after federal approval for personal or horticultural plantings. Methods to accelerate pollen production and outcrossing are discussed.

The chicken hypersensitive site-4 insulator cannot fully shield the murine phosphoglycerate kinase-1 promoter from integration site effects in transgenic mice.

Differential expression of transgenes in transgenic animals is one of the main drawbacks of pronuclear injection. To overwhelm this issue, the genetic constructs are equipped with insulators. In this study, the consensus of exerting chicken hypersensitive site-4 (cHS4) insulator was examined on the shield of phosphoglycerate kinase-1 (Pgk-1) promoter from the surrounding chromatin in transgenic mice. The PGK-EGFP cassette was flanked by insertion of three copies of the cHS4 insulators. Mouse zygotes' microinjection by the constructed cassette was resulted in the birth of nine transgenic founders (F0). Copy-number-dependent expression of the EGFP was investigated in the transgenic F1 offspring by fluorometry and real-time PCR. They showed no correlation between the expression level of transgene and gene copy number among the transgenic lines. Moreover, dissection of the EGFP-expressing mice revealed heterogeneous expression of the EGFP in the different organs. In conclusion, for the first time, these findings indicated that the cHS4 sequence is not a perfect insulator to fully protect the Pgk-1 promoter from the side effects of integration site in transgenic mice and it needs probably to some additional elements of the cHS4 locus to reach this goal.

Transgenic pigs expressing β-xylanase in the parotid gland improve nutrient utilization.

Xylan is one of the main anti-nutritional factors in pig's feed. Although supplementation of β-xylanase in diet can improve the utilization of nutrients in animals, it is limited by feed cost, manufacturing process and storage stability. To determine whether the expression of endogenous β-xylanase gene xynB in vivo can improve digestibility of dietary xylan and absorption of nutrients, we produced transgenic pigs which express the xynB from Aspergillus Niger CGMCC1067 in the parotid glandvia nuclear transfer. In four live transgenic founders, β-xylanase activities in the saliva were 0.74, 0.59, 0.37 and 0.24 U/mL, respectively. Compared with non-transgenic pigs, the content of crude protein (CP) in feces reduced by 15.5% (P < 0.05). Furthermore, in 100 of the 271 F1 pigs the xynB gene was detectable. The digestibility of gross energy and CP in F1 transgenic pigs were increased by 5% and 22%, respectively, with the CP content in feces decreased by 6.4%. Taken together, our study showed that thetransgenic pigs producing β-xylanase from parotid gland can reduce the anti-nutritional effect in animal diet and improve the utilization of nutrients.

94 Impact of number of embryos transferred on the number of offspring produced in a commercial transgenic founder production operation

The production of transgenic founder dairy goats (cross-bred Saanens, Alpines, Toggenburgs, and Nubians) involves the collection, microinjection, and transfer of numerous embryos into suitable recipient goats to ultimately produce a transgenic founder(s). The objective of this study was to determine the most efficient number of microinjection embryos to transfer to suitable recipients for transgenic founder generation. This is critically important in a commercial production program, as it impacts the goal for the number of embryos collected from donors, number of recipients utilised, and, hence, the overall number of surgical procedures being performed. The entire embryo collection, transfer, and founder-generation process is continuously being evaluated for ways to become more efficient in producing transgenic animals. During LFB USA’s commercial founder-production campaigns over the years (1997-2017), pronuclear microinjection was performed and 3, 4, or 5 embryos were transferred to female goat recipients. The recipients were synchronized using a progesterone implant on Day 0, a prostaglandin injection at Day 7, an injection of 300-500IU of pregnant mare serum gonadotropin on Day 13, progesterone implant removal on Day 14, and surgical transfer of pronuclear microinjected 1- or 2-cell embryos into the oviduct on Day 17. The individual totals and calculation for offspring per embryos transferred was compared for 3, 4, and 5 embryos transferred per recipient and was determined to be (1659/8637) 0.19, (912/4548) 0.20, and (112/675) 0.17, respectively. These embryo efficacy ratios were not significantly different (P&amp;gt;0.05) using the Wald Chi-squared test under logistic regression, and suggests that the number of offspring born is not impacted by number of embryos transferred. Seasonality was also evaluated in this production environment located in North America, with in-season being considered September to December and out-of-season being January to July. Nulliparous recipients during in-season (September to December) embryo transfer operations produced a significant difference, with totals and calculation for (offspring per embryo transferred) of (470/2346) 0.20, (260/1088) 0.24, and (23/190) 0.12 for 3, 4, and 5 embryo transfers, respectively (Table 1). This data indicates that when using nulliparous recipients during the in-season, transferring 4 embryos is optimal for offspring produced. Table 1.Comparison of the individual totals and the calculation of (offspring/embryo) by parity and season

194 Superovulation response does not affect embryo development of pronuclear microinjected embryos in the goat

Superovulation of donor animals is essential in the production of transgenic founder goats generated through microinjection. There can be a marked variable response to the exogenous hormones used for superovulation. The objective of this study was to examine how the superovulatory response of individual goats affected the ability of the fertilized, microinjected embryos to develop into offspring. The donors were superovulated using a progesterone implant on Day 0, a prostaglandin injection at Day 7, 2 injections ~12h apart of 32 to 36mg of FSH on Day 12 to 15, progesterone implant removal on Day 14, bred by intact bucks several times starting on Day 15 to 16, an injection of 50μg of gonadotropin-releasing hormone, and surgical collection of 1- to 2-cell embryos from retrograde flushing of the oviduct on Day 17 (~24-48 h, 1-2 days after breeding). Surgical collection allows for an accurate ovulation point (OP) count before the oviduct being retrograde flushed and ova collected and counted. Data from donor animals were grouped by superovulatory response based on OP counts of 1-10, 11-20, 21-30, or &amp;gt;30. The number of donors that contributed per group were 130, 280, 175, and 52, respectively. The recovery rate was 76, 72, 68, and 62%, respectively. After collection, ova were viewed under a dissecting microscope and assessed for fertilization by identifying pronuclei, and 1 pronucleus was microinjected. The fertilization rate was 47, 52, 51, and 56%, respectively. The survivability rate after microinjection was 80, 76, 75, and 76%, respectively. Surviving embryos were transferred (3-5) into recipient goats following a 2- to 6-h in vitro culture (as 1- to 2-cell embryos), allowing for a suitable period to assess viability post-injection. Further in vitro development rates were not assessed because of the short timeframe the embryos stayed in culture. The conception rates were 71, 56, 65, and 53%, respectively, and abortion rates were 23, 10, 14, and 9%, respectively. As some recipients received embryos from multiple donors, this data could not be included in the analysis as identifying which offspring were from the corresponding embryo group could not be confirmed. Data were analysed using SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). The Wald chi square test under linear regression was used to analyse the number of offspring produced per embryo transferred. No significant differences were found between groups (all P-values were&amp;gt;0.05). This analysis indicated that the range of superovulation response does not affect the developmental competence of the pronuclear microinjected embryo or the ability to produce viable offspring. Table 1.Comparison of the donor ovulation counts, number of embryos transferred, offspring produced and overall efficiency

Keratin 4 regulates the development of human white sponge nevus.

The aim of this study was to investigate the roles of keratin 4 (KRT4) gene in the development of human white sponge nevus (WSN). Transgenic mice were created using the microinjection method with pcDNA3.1 vectors expressing KRT4 wild-type (WT) gene and E520K mutation. Polymerase chain reaction (PCR) and Western blotting were used to identify the genotype of transgenic founders and their filial generations. Expression of KRT4 in mouse oral mucosa was characterized by immunohistochemistry (IHC), and the whole epithelium layer of transgenic mice was observed using transmission electron microscope (TEM). The positive rate of KRT4 transgenic mice in F1 generation was 45.5%. Expression level of KRT4 protein was significantly higher in 2-month-old transgenic mice than WT mice. Furthermore, all the epithelial lamina of 3-month-old transgenic mice showed reduced staining of KRT4. The surface and spinous layers were full of hyalocytes and bubble cells, which are similar to the clinical symptoms of WSN. For the ultrastructure, both tonofilaments and Odland bodies increased. Our study indicated the mutated KRT4 gene may play important roles in the pathogenesis of WSN.

Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

BackgroundMedaka (Oryzias latipes) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed.ResultsWe report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ). Donor plasmid containing a heat-shock promoter and a reporter gene was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. Broad transgene expression in the expression domain of a target gene was observed in approximately 25% of injected embryos. By raising these animals, we established stable knock-in transgenic fish with several different constructs for five genetic loci, obtaining transgenic founders at efficiencies of > 50% for all five loci. Further, we show that the method is useful for obtaining mutant alleles. In the experiments where transgene integrations were targeted between the transcription start site and the initiation methionine, the resultant transgenic fish became mutant alleles.ConclusionWith its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in via NHEJ will become a standard method for the generation of transgenic and mutant medaka.

Generation of transgenic goats by pronuclear microinjection: a retrospective analysis of a commercial operation (1995-2012).

Production of transgenic founder goats involves introducing and stably integrating an engineered piece of DNA into the genome of the animal. At LFB USA, the ultimate use of these transgenic goats is for the production of recombinant human protein therapeutics in the milk of these dairy animals. The transgene or construct typically links a milk protein specific promoter sequence, the coding sequence for the gene of interest, and the necessary downstream regulatory sequences thereby directing expression of the recombinant protein in the milk during the lactation period. Over the time period indicated (1995-2012), pronuclear microinjection was used in a number of programs to insert transgenes into 18,120, 1- or 2- cell stage fertilized embryos. These embryos were transferred into 4180 synchronized recipient females with 1934 (47%) recipients becoming pregnant, 2594 offspring generated, and a 109 (4.2%) of those offspring determined to be transgenic. Even with new and improving genome editing tools now available, pronuclear microinjection is still the predominant and proven technology used in this commercial setting supporting regulatory filings and market authorizations when producing founder transgenic animals with large transgenes (>10kb) such as those necessary for directing monoclonal antibody production in milk.

Effects of pituitary-specific overexpression of FSH\u03b1/\u03b2 on reproductive traits in transgenic boars

BackgroundFollicle-stimulating hormone (FSH) is a gonadotropin synthesized and secreted by the pituitary gland. FSH stimulates follicle development and maturation in females. It also plays an important role in spermatogenesis in males, including humans and mice. However, the effects of FSH on male pigs are largely unknown. In this study, we generated transgenic pigs to investigate the effects of FSHα/β overexpression on reproductive traits in boars.ResultsAfter five transgenic F0 founders were crossed with wide-type pigs, 193 F1 animals were obtained. Of these, 96 were confirmed as transgenic. FSHα and FSHβ mRNAs were detected only in pituitary tissue. Transgenic boars exhibited significantly higher levels of FSHα and FSHβ mRNA, serum FSH, and serum testosterone, compared to full-sib non-transgenic boars. Significant increases in testis weight, vas deferens diameter, seminiferous tubule diameter, and the number of Leydig cells were observed, suggesting that the exogenous FSHα/β affects reproductive traits. Finally, transgenic and non-transgenic boars had similar growth performance and biochemical profiles.ConclusionsPituitary-specific overexpression of FSHα/β genes is likely to impact reproductive traits positively, as indicated by enhancements in serum testosterone level, testis weight, the development of vas deferens, seminiferous tubules, and Leydig cells in transgenic boars. A high level of serum FSH induces secretion of serum testosterone, possibly by boosting the number of Leydig cells, which presumably increases the libido and the frequency of sexual activity in transgenic boars. Our study provides a preliminary foundation for the genetic improvement of reproductive traits in male pigs.