Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by &lt;i&gt;In Vitro&lt;/i&gt; Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Umeyama, Kazuhiro; Honda, Kasumi; Matsunari, Hitomi; Nakano, K.; Hidaka, Tatsuro; Sekiguchi, Keito; Mochizuki, Hironori; Takeuchi, Yasuhiro; Fujiwara, Tsukasa; Watanabe, Masaru; Nagaya, Masaki; Nagashima, Hiroshi

doi:10.1262/jrd.2013-069

Cited by 11 publications

(6 citation statements)

References 15 publications

(22 reference statements)

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“…Thirty-percent of oocytes were successfully penetrated (15/50), and the majority showed normal fertilization (86.7%, 13/15). The resulting blastocyst rate as well as the number of cells per blastocyst was within the range reported in previous IVF studies [ 26 , 27 ] ( S1 Fig ).…”

Section: Resultssupporting

confidence: 86%

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs

Bähr¹,

Käser

Kemter³

et al. 2016

PLoS ONE

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We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action.

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Section: Resultssupporting

confidence: 86%

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs

Bähr¹,

Käser

Kemter³

et al. 2016

PLoS ONE

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“…Porcine zygotes (German landrace background) were produced in vitro as described previously [22] , and Cas9 mRNA (50 ng/μL) and sgRNA (100 ng/μL) was injected into their cytoplasm 8.5–9.5 h after in vitro fertilization. Recipient gilts were synchronized in the estrous cycle by oral administration of 4 mL Altrenogest (Regumate ® ; MSD Animal Health) for 15 days, followed by intramuscular injection of 750 IU ECG (Intergonan ® ; MSD Animal Health) and 750 IU HCG (Ovogest ® ; MSD Animal Health) after an additional 24 and 104 h, respectively.…”

Section: Methodsmentioning

confidence: 99%

Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver

Hinrichs

Kessler

Kurome³

et al. 2018

Molecular Metabolism

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ObjectiveLaron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized.MethodsCRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue.ResultsGHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation of JAK2 was significantly increased, possibly due to the increased serum leptin levels and increased hepatic leptin receptor expression and activation in GHR-KO pigs. In addition, increased mTOR phosphorylation was observed in GHR-KO liver samples, and phosphorylation studies of downstream substrates suggested the activation of mainly mTOR complex 2.ConclusionGHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials.

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“…The IVF method has been described previously [ 29 ]. Briefly, porcine ovaries were collected at local abattoirs, and in vitro matured oocytes with expanded cumulus cells were used for IVF.…”

Section: Methodsmentioning

confidence: 99%

Establishment of DNA methylation patterns of the <i>Fibrillin1</i> (<i>FBN1</i>) gene in porcine embryos and tissues

Arai

Umeyama

Takeuchi

et al. 2017

Journal of Reproduction and Development

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DNA methylation in transcriptional regulatory regions is crucial for gene expression. The DNA methylation status of the edges of CpG islands, called CpG island shore, is involved in tissue/cell-type-specific gene expression. Haploinsufficiency diseases are caused by inheritance of one mutated null allele and are classified as autosomal dominant. However, in the same pedigree, phenotypic variances are observed despite the inheritance of the identical mutated null allele, including Fibrillin1 (FBN1), which is responsible for development of the haploinsufficient Marfan disease. In this study, we examined the relationship between gene expression and DNA methylation patterns of the FBN1 CpG island shore focusing on transcriptionally active hypomethylated alleles (Hypo-alleles). No difference in the DNA methylation level of FBN1 CpG island shore was observed in porcine fetal fibroblast (PFF) and the liver, whereas FBN1 expression was higher in PFF than in the liver. However, Hypo-allele ratio of the FBN1 CpG island shore in PFF was higher than that in the liver, indicating that Hypo-allele ratio of the FBN1 CpG island shore likely correlated with FBN1 expression level. In addition, oocyte-derived DNA hypermethylation in preimplantation embryos was erased until the blastocyst stage, and re-methylation of the FBN1 CpG island shore was observed with prolonged in vitro culture of blastocysts. These results suggest that the establishment of the DNA methylation pattern within the FBN1 CpG island shore occurs after the blastocyst stage, likely during organogenesis. In conclusion, Hypo-allele ratios of the FBN1 CpG island shore correlated with FBN1 expression levels in porcine tissues.

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Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by <i>In Vitro</i> Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Cited by 11 publications

References 15 publications

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs

Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver

Establishment of DNA methylation patterns of the <i>Fibrillin1</i> (<i>FBN1</i>) gene in porcine embryos and tissues

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