Conceptus Estrogen and prostaglandins provide the maternal recognition of pregnancy signal to prevent Luteolysis during early pregnancy in the pig†.

Conceptus estrogens and prostaglandins have long been considered the primary signals for maternal recognition of pregnancy (MRP) in the pig. However, loss-of-function studies targeting conceptus aromatase genes (CYP19A1 and CYP19A2) and prostaglandin-endoperoxide synthase 2 (PTGS2) indicated that conceptuses can not only signal MRP without estrogens or prostaglandins but can maintain early pregnancy. However, complete loss of estrogen production leads to abortion after day 25 of gestation. Although neither conceptus estrogens nor prostaglandins had a significant effect on early maintenance of CL function alone, the two conceptus factors have a biological relationship. To investigate the role that both conceptus estrogens and prostaglandins have on MRP and maintenance of pregnancy, a triple loss-of function model (TKO) was generated for conceptus CYP19A1, CYP19A2 and PTGS2. In addition, a conceptus CYP19A2-/- model (A2KO) was established to determine the role of placental estrogen during later pregnancy. Estrogen and prostaglandin synthesis were greatly reduced in TKO conceptuses which resulted in a failure to inhibit luteolysis after day 15 of pregnancy despite the presence of conceptuses in the uterine lumen. However, A2KO placentae not only maintained functional CL but were able to maintain pregnancy to day 32 of gestation. Despite the loss of placental CYP19A2 expression, the allantois fluid content of estrogen was not affected as the placenta compensated by expressing CYP19A1 and CYP19A3, which are normally absent in controls. Results suggest conceptuses can signal MRP through production of conceptus PGE or stimulating PGE synthesis from the endometrium through conceptus estrogen. Failure of conceptuses to produce both factors results in failure of MRP and loss of pregnancy.

Porcine-specific expression of the three functional CYP19 paralogs in early conceptus, placenta, and gonads.

In brief: Aromatase catalyzes the synthesis of estrogens and has been shown to have an important role during the establishment of pregnancy in the pig. This study confirmed the differential expression of the three aromatase isoforms. Abstract: Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal-placental development. Cyp19a1 and Cyp19a3 expressions were detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both the conceptuses and the placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of day 21 pregnant females, as well as days 12 and 17 pseudopregnant females, we did not detect the expression of Cyp19a1, Cyp19a2, or Cyp19a3. In our study, we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling the secretion of estrogen from the conceptus and the placenta.

Gene editing provides a tool to investigate genes involved in reproduction of pigs.

CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.

Chloride channel accessory 1 gene deficiency causes selective loss of mucus production in a new pig model.

Morbidity and mortality of respiratory diseases are linked to airway obstruction by mucus but there are still no specific, safe, and effective drugs to correct this phenotype. The need for better treatment requires a new understanding of the basis for mucus production. In that regard, studies of human airway epithelial cells in primary culture show that a mucin granule constituent known as chloride channel accessory 1 (CLCA1) is required for inducible expression of the inflammatory mucin MUC5AC in response to potent type 2 cytokines. However, it remained uncertain whether CLCLA1 is necessary for mucus production in vivo. Conventional approaches to functional biology using targeted gene knockout were difficult due to the functional redundancy of additional Clca genes in mice not found in humans. We reasoned that CLCA1 function might be better addressed in pigs that maintain the same four-member CLCA gene locus and the corresponding mucosal and submucosal populations of mucous cells found in humans. Here we develop to our knowledge the first CLCA1-gene-deficient (CLCA1-/-) pig and show that these animals exhibit loss of MUC5AC+ mucous cells throughout the airway mucosa of the lung without affecting comparable cells in the tracheal mucosa or MUC5B+ mucous cells in submucosal glands. Similarly, CLCA1-/- pigs exhibit loss of MUC5AC+ mucous cells in the intestinal mucosa without affecting MUC2+ mucous cells. These data establish CLCA1 function for controlling MUC5AC expression as a marker of mucus production and provide a new animal model to study mucus production at respiratory and intestinal sites.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Conceptus interferon gamma is essential for establishment of pregnancy in the pig†.

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG-/- conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG-/- conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG-/- embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs.

Disrupting porcine glutaminase does not block preimplantation development and elongation nor decrease mTORC1 activation in conceptuses†.

Elongation of pig conceptuses is a dynamic process, requiring adequate nutrient provisions. Glutamine is used as an energy substrate and is involved in the activation of mechanistic target of rapamycin complex 1 (mTORC1) during porcine preimplantation development. However, the roles of glutamine have not been extensively studied past the blastocyst stage. Therefore, the objective of the current study was to determine if glutaminase (GLS), which is the rate-limiting enzyme in glutamine metabolism, was necessary for conceptus elongation to proceed and was involved in mTORC1 activation. The CRISPR/Cas9 system was used to induce loss-of-function mutations in the GLS gene of porcine fetal fibroblasts. Wild type (GLS+/+) and knockout (GLS-/-) fibroblasts were used as donor cells for somatic cell nuclear transfer, and GLS+/+ and GLS-/- blastocyst-stage embryos were transferred into surrogates. On day 14 of gestation, GLS+/+ conceptuses primarily demonstrated filamentous morphologies, and GLS-/- conceptuses exhibited spherical, ovoid, tubular, and filamentous morphologies. Thus, GLS-/- embryos were able to elongate despite the absence of GLS protein and minimal enzyme activity. Furthermore, spherical GLS-/- conceptuses had increased abundance of transcripts related to glutamine and glutamate metabolism and transport compared to filamentous conceptuses of either genotype. Differences in phosphorylation of mTORC1 components and targets were not detected regarding conceptus genotype or morphology, but abundance of two transcriptional targets of mTORC1, cyclin D1, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha was increased in spherical conceptuses. Therefore, porcine GLS is not essential for conceptus elongation and is not required for mTORC1 activation at this developmental timepoint.

Gene editing to investigate the role of conceptus factors in the establishment of pregnancy in the pig.

Development of viviparity in mammals requires that the placenta evolves as an intermediate interface between the fetus and maternal uterus. In addition to the retention of the fetus and secretion of nutrients to support growth and development to term, it is essential that viviparous species modify or inhibit the maternal immune system from recognizing the semi-allogeneic fetus. Following blastocyst hatching from its zona pellucida, trophoblast differentiation provides the initial communication to the maternal endometrium to regulate maintenance of progesterone production from the corpus luteum and biological pathways in uterine and conceptus development necessary in the establishment and maintenance of pregnancy. Many conceptus factors have been proposed to serve in the establishment and maintenance of pregnancy. CRISPR-Cas9 gene-editing technology provides a specific and efficient method to generate animal models to perform loss-of-function studies to investigate the role of specific conceptus factors. The utilization of CRISPR-Cas9 gene editing has provided a direct approach to investigate the specific role of conceptus factors in the development and establishment of pregnancy in the pig. This technology has helped address a number of questions concerning peri-implantation development and has altered our understanding of maternal recognition and maintenance of pregnancy in the pig.

Ablation of conceptus PTGS2 expression does not alter early conceptus development and establishment of pregnancy in the pig†.

Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2-/- blastocysts on day 7. PTGS2+/+ and PTGS2-/- blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3 h to determine PG production. Conceptus release of total PG, prostaglandin F2⍺ (PGF2α), and PGE in culture media was lower with PTGS2-/- conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2-/- conceptus surrogates allowed to continue pregnancy were maintained beyond 30 days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig.

New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig†.

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia.

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1-3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere-like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.

Resistance to coronavirus infection in amino peptidase N-deficient pigs.

The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.

Using a keratinase to degrade chicken feathers for improved extraction of glucocorticoids.

Stress in animals is a concern in conservation breeding programs and livestock production facilities. The biological stress response is mediated by the release of glucocorticoids, which can suppress reproduction, growth, and immunity if recurrently activated. Feathers can be used to extract and monitor concentrations of corticosterone, a primary glucocorticoid in birds. However, current techniques for extracting feather corticosterone present challenges, including difficulty assessing extraction efficiency or hormone recovery, inconsistent extraction across feather lengths or pieces, and several uncertainties regarding the mechanisms of hormone deposition into feathers. To overcome such challenges and to provide tools useful for facilitating conservation breeding and livestock production, we developed and validated an alternative procedure for extracting feather glucocorticoids. We first developed a protocol to enzymatically digest the protein matrix of feathers using a keratinase, such that non-protein analytes could be isolated by organic extraction. We then developed an extraction protocol and evaluated techniques by measuring extraction efficiency and by testing parallelism and hormone recovery (accuracy) using radioimmunoassay. Our results demonstrated high and consistent extraction efficiency, as well as high accuracy and reliable parallelism to a standard curve upon measurement of corticosterone concentrations from extracts. By dissolving feather material into solution prior to extraction, we were able to replicate hormone deposition into the feather matrix and ensure consistent extraction across feathers. This work provides additional support for the validity and practicality of extracting glucocorticoids from feathers. Our extraction protocol is likely to extend to other applications as well, including the isolation of numerous non-protein analytes from various keratinized tissues.

Replacement of Porcine CD163 Scavenger Receptor Cysteine-Rich Domain 5 with a CD163-Like Homolog Confers Resistance of Pigs to Genotype 1 but Not Genotype 2 Porcine Reproductive and Respiratory Syndrome Virus.

CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection of CD163-modified pigs with representative type 1 and type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163. IMPORTANCE: Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions.

Pharmacologic treatment of donor cells induced to have a Warburg effect-like metabolism does not alter embryonic development in vitro or survival during early gestation when used in somatic cell nuclear transfer in pigs.

Somatic cell nuclear transfer is a valuable technique for the generation of genetically engineered animals, however, the efficiency of cloning in mammalian species is low (1-3%). Differentiated somatic cells commonly used in nuclear transfer utilize the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively the metabolism of somatic cells contrasts that of the cells within the early embryos which predominately use glycolysis. Early embryos (prior to implantation) are evidenced to exhibit characteristics of a Warburg Effect (WE)-like metabolism. We hypothesized that pharmacologically driven fibroblast cells can become more blastomere-like and result in improved in vitro embryonic development after SCNT. The goals were to determine if subsequent in vitro embryo development is impacted by (1) cloning pharmacologically treated donor cells pushed to have a WE-like metabolism or (2) culturing non-treated donor clones with pharmaceuticals used to push a WE-like metabolism. Additionally, we investigated early gestational survival of the donor-treated clone embryos. Here we demonstrate that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs. Furthermore, these experiments demonstrate that early embryos (or at least in vitro produced embryos) have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos. Lastly, we show that survival in early gestation was not different between clones from pharmacologically induced WE-like donor cells and controls.

Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs.

Genetically engineered pigs are often created with a targeting vector that contains a loxP flanked selectable marker like neomycin. The Cre-loxP recombinase system can be used to remove the selectable marker gene from the resulting offspring or cell line. Here is described a new method to remove a loxP flanked neomycin cassette by direct zygote injection of an mRNA encoding Cre recombinase. The optimal concentration of mRNA was determined to be 10 ng/µL when compared to 2 and 100 ng/µL (P < 0.0001). Development to the blastocyst stage was 14.1% after zygote injection with 10 ng/µL. This method successfully removed the neomycin cassette in 81.9% of injected in vitro derived embryos; which was significantly higher than the control (P < 0.0001). Embryo transfer resulted in the birth of one live piglet with a Cre deleted neomycin cassette. The new method described can be used to efficiently remove selectable markers in genetically engineered animals without the need for long term cell culture and subsequent somatic cell nuclear transfer.

Genome-editing technologies to improve research, reproduction, and production in pigs.

The ability to directly manipulate the pig genome through genetic engineering has been available to the research community for over three decades. This technology has progressed from the random insertion of foreign DNA, via a variety of techniques (pronuclear microinjection, sperm mediated gene transfer, and integration of mobile genetic elements), to manipulation of endogenous genes, via homologous recombination in somatic cells followed by somatic cell nuclear transfer. Over the last few years, designer nucleases facilitated the development of techniques that provide efficient ways to introduce foreign DNA or to modify endogenous genes in eggs, zygotes, or somatic cells. Together, these genome-editing technologies have essentially removed the obstacles to gene manipulation in swine. Although the regulatory environment is still unclear for agricultural applications, genetic engineering of pigs will continue to advance biomedicine and biology. In addition, genetic engineering is now sufficiently simple and efficient that agricultural research can now ask basic and applied questions that are not hampered by limited funding.

Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs.

The CRISPR/Cas9 genome editing tool has increased the efficiency of creating genetically modified pigs for use as biomedical or agricultural models. The objectives were to determine if DNA editing resulted in a delay in development to the blastocyst stage or in a skewing of the sex ratio. Six DNA templates (gBlocks) that were designed to express guide RNAs that target the transmembrane protease, serine S1, member 2 (TMPRSS2) gene were in vitro transcribed. Pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. Blastocysts were collected as they formed on days 5, 6 or 7. PCR was performed to determine genotype and sex of each embryo. Separately, embryos were surgically transferred into recipient gilts on day 4 of estrus. The rate of blastocyst development was not significantly different between CRISPR injection embryos or the non-injected controls at day 5, 6 or 7 (p = 0.36, 0.09, 0.63, respectively). Injection of three CRISPR sets of guides resulted in a detectable INDEL in 92-100 % of the embryos analyzed. There was not a difference in the number of edits or sex ratio of male to female embryos when compared between days 5, 6 and 7 to the controls (p > 0.22, >0.85). There were 12 resulting piglets and all 12 had biallelic edits of TMRPSS2. Zygote injection with CRISPR/Cas9 continues to be a highly efficient tool to genetically modify pig embryos.

Transcriptomic differences in intra-abdominal adipose tissue in extremely obese adolescents with different stages of NAFLD.

Mechanisms responsible for progression of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH) remain poorly defined. To examine the potential contribution of adipose tissue to NAFLD progression, we performed a complete transcriptomic analysis using RNA sequencing (RNA-Seq) on intra-abdominal adipose tissue (IAT) from severely obese adolescents [Mage 16.9 ± 0.4 yr, body mass index (BMI) z-score 2.7 ± 0.1] undergoing bariatric surgery and liver biopsy categorized into three groups: no steatosis (normal, n = 8), steatosis only (n = 13), or NASH (n = 10) by liver histology. Age, body weight, and BMI did not differ among groups, but subjects with NASH were more insulin resistant (increased homeostatic model assessment/insulin resistance, P < 0.05 vs. other groups). RNA-Seq revealed 175 up- and 492 downregulated mRNA transcripts (≥±1.5-fold, false discovery rate <0.10) in IAT between NASH vs. Normal, with "mitochondrial dysfunction, P = 4.19E-7" being the top regulated canonical pathway identified by Ingenuity Pathway Analysis; only 19 mRNA transcripts were up- and 148 downregulated when comparing Steatosis vs. Normal, with suppression of "EIF2 signaling, P = 1.79E-27" being the top regulated pathway indicating increased cellular stress. A comparison of IAT between NASH vs. Steatosis found 515 up- and 175 downregulated genes, with "antigen presentation, P = 6.03E-18" being the top regulated canonical pathway and "inflammatory response" the top diseases and disorders function. Unique transcriptomic differences exist in IAT from severely obese adolescents with distinct stages of NAFLD, providing an important resource for identifying potential novel therapeutic targets for childhood NASH.

Genetic engineering of mammals.

Historically, genetic engineering for mammalian reproductive questions has been accomplished primarily in the mouse. However, all the genetic manipulations that can be done in the mouse can now be accomplished in most domesticated mammals. Random integration of transgenes, homologous recombination and gene editing are now routine for several mammalian species. For livestock, queries related to fertility can be asked directly for the species in question, without a need for a mouse model. For human clinical concerns, the most appropriate model should be selected based on physiology, anatomy, or even size. The mouse will continue to be a useful genetically engineered model. However, other species are now amenable to the full range of genetic manipulations and should be considered as possible models for human conditions when appropriate.