Search

Purpose The mechanism of cardioprotection by Kv7.1-5 (KCNQ1-5) channels inhibition by XE991 is unclear. We examined the impact of administration time on the cardioprotective efficacy of XE991, the involvement of key pro-survival kinases, and the importance of the Kv7 subchannels. Methods Isolated perfused rat hearts were divided into five groups: 1) vehicle, 2) pre-, 3) post- or 4) pre- and post-ischemic administration of XE991 or 5) chromanol 293B (Kv7.1 inhibitor) followed by infarct size quantification. HL-1 cells undergoing simulated ischemia/reperfusion were exposed to either a) vehicle, b) pre-, c) per-, d) post-ischemic administration of XE991 or pre-, per- and post-ischemic administration of e) XE991, f) Chromanol 293B or g) HMR1556 (Kv7.1 inhibitor). HL-1 cell injury was evaluated by propidium iodide/Hoechst staining. Pro-survival kinase activation of Akt, Erk and STAT3 in XE991-mediated HL-1 cell protection was evaluated using phosphokinase inhibitors. Kv7 subtype expression was examined by RT-PCR and qPCR. Results XE991, but not Chromanol 293B, reduced infarct size and improved hemodynamic recovery in all isolated heart groups. XE991 protected HL-1 cells when administered during simulated ischemia. Minor activation of the survival kinases was observed in cells exposed to XE991 but pharmacological inhibition of kinase activation did not reduce XE991-mediated protection. Kv7 subchannels 1-5 were all present in rat hearts but predominately Kv7.1 and Kv7.4 were present in HL-1 cells and selective Kv7.1 did not reduce ischemia/reperfusion injury. Conclusion The cardioprotective efficacy of XE991 seems to depend on its presence during ischemia and early reperfusion and do not rely on RISK (p-Akt and p-Erk) and SAFE (p-STAT3) pathway activation. The protective effect of XE991 seems mainly mediated through the Kv7.4 subchannel.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder for which no cure exists. The disease is characterized by premature aging and inevitable death in adolescence due to cardiovascular complications. Most HGPS patients carry a heterozygous de novo LMNA c.1824C > T mutation, which provokes the expression of a dominant-negative mutant protein called progerin. Therapies proven effective in HGPS-like mouse models have yielded only modest benefit in HGPS clinical trials. To overcome the gap between HGPS mouse models and patients, we have generated by CRISPR-Cas9 gene editing the first large animal model for HGPS, a knockin heterozygous LMNA c.1824C > T Yucatan minipig. Like HGPS patients, HGPS minipigs endogenously co-express progerin and normal lamin A/C, and exhibit severe growth retardation, lipodystrophy, skin and bone alterations, cardiovascular disease, and die around puberty. Remarkably, the HGPS minipigs recapitulate critical cardiovascular alterations seen in patients, such as left ventricular diastolic dysfunction, altered cardiac electrical activity, and loss of vascular smooth muscle cells. Our analysis also revealed reduced myocardial perfusion due to microvascular damage and myocardial interstitial fibrosis, previously undescribed readouts potentially useful for monitoring disease progression in patients. The HGPS minipigs provide an appropriate preclinical model in which to test human-size interventional devices and optimize candidate therapies before advancing to clinical trials, thus accelerating the development of effective applications for HGPS patients. This project was mainly supported by an Established Investigator Award from the Progeria Research Foundation (2014-52), and from the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU), and the European Regional Development Fund (FEDER, “A way to build Europe”) (SAF2016-79490-R, CB16/11/00405). Ana Barettino has a predoctoral contract from MCIU (BES-2017-079705). Work at Universidad de Murcia is supported by Fundación Seneca-Agencia de Ciencia y Tecnología de la Región de Murcia (20040/GERM/16). The CNIC is supported by the MCIU and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Sí

Coronary heart disease and ischaemic stroke caused by atherosclerosis are leading causes of illness and death worldwide. Small animal models have provided insight into the fundamental mechanisms driving early atherosclerosis, but it is increasingly clear that new strategies and research tools are needed to translate these discoveries into improved prevention and treatment of symptomatic atherosclerosis in humans. Key challenges include better understanding of processes in late atherosclerosis, factors affecting atherosclerosis in the coronary bed, and the development of reliable imaging biomarker tools for risk stratification and monitoring of drug effects in humans. Efficient large animal models of atherosclerosis may help tackle these problems. Recent years have seen tremendous advances in gene-editing tools for large animals. This has made it possible to create gene-modified minipigs that develop atherosclerosis with many similarities to humans in terms of predilection for lesion sites and histopathology. Together with existing porcine models of atherosclerosis that are based on spontaneous mutations or severe diabetes, such models open new avenues for translational research in atherosclerosis. In this review, we discuss the merits of different animal models of atherosclerosis and give examples of important research problems where porcine models could prove pivotal for progress.

Visual Abstract, Highlights • APOE-deficient Yucatan minipigs were created by recombinant adeno-associated virus mediated gene targeting in porcine fibroblasts followed by somatic cell nuclear transfer. • APOE−/− minipigs displayed increased plasma cholesterol and accumulation of APOB48-containing chylomicron remnants on low fat-diet, which was significantly accentuated upon feeding a high-fat, high-cholesterol diet. • APOE−/− minipigs showed accelerated progressive atherosclerosis but not xanthoma formation indicating that remnant lipoproteinemia does not induce early lesions but is atherogenic in pre-existing atherosclerosis., Summary Deficiency of apolipoprotein E (APOE) causes familial dysbetalipoproteinemia in humans resulting in a higher risk of atherosclerotic disease. In mice, APOE deficiency results in a severe atherosclerosis phenotype, but it is unknown to what extent this is unique to mice. In this study, APOE was targeted in Yucatan minipigs. APOE−/− minipigs displayed increased plasma cholesterol and accumulation of apolipoprotein B-48–containing chylomicron remnants on low-fat diet, which was significantly accentuated upon feeding a high-fat, high-cholesterol diet. APOE−/− minipigs displayed accelerated progressive atherosclerosis but not xanthoma formation. This indicates that remnant lipoproteinemia does not induce early lesions but is atherogenic in pre-existing atherosclerosis.

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells.

Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applicability to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer (SCNT), this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.

During the last two decades, pigs have been used to develop some of the most important large animal models for biomedical research. Advances in pig genome research, genetic modification (GM) of primary pig cells and pig cloning by nuclear transfer, have facilitated the generation of GM pigs for xenotransplantation and various human diseases. This review summarizes the key technologies used for generating GM pigs, including pronuclear microinjection, sperm-mediated gene transfer, somatic cell nuclear transfer by traditional cloning, and somatic cell nuclear transfer by handmade cloning. Broadly used genetic engineering tools for porcine cells are also discussed. We also summarize the GM pig models that have been generated for xenotransplantation and human disease processes, including neurodegenerative diseases, cardiovascular diseases, eye diseases, bone diseases, cancers and epidermal skin diseases, diabetes mellitus, cystic fibrosis, and inherited metabolic diseases. Thus, this review provides an overview of the progress in GM pig research over the last two decades and perspectives for future development.

In this study, we compared the gene targeting efficiencies of two rAAV-BRCA1 KO targeting constructs in Yucatan and Göttingen minipig fibroblasts. The homology arms of the constructs consisted exclusively of exonic sequences amplified by PCR from Yucatan genomic DNA. The sequences were identical to those of the reference porcine genome of a Duroc sow (Ensembl Susscrofa 9) and the BRCA1 gene of the Landrace breed (NCBI acc. no. AB271921). Surprisingly, we found that the very efficient gene targeting observed for Yucatan fibroblasts (35% targeting efficiency) was completely absent using either of the two constructs in Göttingen fibroblasts. Sequencing of the relevant BRCA1 exon 11 region (~2 kb) in the Göttingen minipig revealed three single nucleotide differences in the sequence targeted by the left homology arm of the construct (0.3% of the bases) and three or seven in the two right homology regions (0.3 or 0.7% of the bases, respectively). Construction of a novel rAAV-BRCA1 targeting vector based on the Göttingen genomic DNA sequence re-established gene targeting although the efficiency was somewhat lower than that observed for Yucatan fibroblasts. These BRCA1 KO Göttingen fibroblast clones have been used as nuclear donor cells for somatic cell nuclear transfer to generate a Göttingen BRCA1 KO pig model as previously done with the Yucatan breed. The present study illustrates that even a few mismatches present in the homology arms of an efficient rAAV-targeting construct can completely abolish gene targeting by homologous recombination emphasizing the importance of using isogenic DNA even for creating targeting constructs consisting of exon sequences only.

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets Udgivelsesdato: September An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0–20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

Introduction: Adjuvant treatment of prostate cancer often involves androgen deprivation therapy (ADT) by orchiectomy (Orx), gonadotropin releasing hormone (GnRH) receptor agonists or GnRH receptor antagonists. Clinical studies of men with preexisting atherosclerotic disease, have indicated that cardiovascular event rates differs between different types of ADT, suggesting that not only testosterone deficiency, but also direct effects of ADT on processes in the atherosclerotic plaque may be involved. Immune cell GnRH receptors have been implicated as potential mediators. In the present study, we compared the effects of common types of ADT on de-novo atherosclerosis in mice. Methods: Chow-fed Apoe-deficient mice (n=121) were allocated to Orx and/or monthly injections with the GnRH receptor agonist leuprolide (0.5 mg s.c.), the GnRH receptor antagonist degarelix (0.5 mg s.c.) or saline starting from 8 weeks of age (n=19-21 in each group). At 26 weeks of age, the burden of atherosclerosis was quantified by en face analysis of the Oil red O-stained aortic arch and in histological sections of the aortic root. Bone marrow-derived macrophages and splenocytes were incubated in the presence of leuprolide (10 -6 mol/L) or degarelix (10 -6 mol/L). Data is shown as mean ± sem, *p Results: All types of ADT augmented hypercholesterolemia and atherosclerosis with no significant differences in the size of the effect (Figure A-B). In Orx mice, the addition of leuprolide, but not degarelix, led to further increases in plasma total cholesterol and in atherosclerosis in the aortic root (D), but not the aortic arch (C). Plaque necrosis was unaffected by ADT. No effects of the drugs on macrophage cytokine secretion or splenocyte proliferation were observed. Conclusions: ADT did not exert differential effects on the development of atherosclerosis in the Apoe-deficient mice. A pro-atherogenic, possibly cholesterol-mediated, effect of leuprolide was seen in Orx mice.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene. Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3' end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN). No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms. Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells.

As part of a strategy to develop somatic gene therapy of epidermolysis bullosa simplex (EBS) we have established patient keratinocytes with expanded lifespan by ectopic expression of the human telomerase gene (hTert). The presence of an active telomerase enzyme was demonstrated by the telomerase repeat amplification protocol (TRAP). The hTert(+) cells have a normal karyotype and the cells have, until now, undergone more than 80 population doublings (PDs) after hTert retroviral transduction while control cells exhibited senescence-associated proliferation arrest after 8 PDs. In organotypic culture the hTert(+) cells are capable of forming a stratified epidermis illustrating their preserved ability to differentiate.

Pigs are increasingly used as genetically modified (GM) animal models for human diseases. Reliable methods to produce GM piglets are needed to produce at least one founder animal that can pass on the transgene to the next generation using conventional reproductive procedures. Somatic cell nuclear transfer (SCNT, “cloning”) is one such method, although it has a low efficiency with up to only 10% of offspring born based on number of cloned embryos transferred (Liu et al. 2014 Reprod. Fertil. Dev. 27, 429-439) and with a high percentage dying in the first days after birth (Schmidt et al. 2015 Theriogenology 84, 1014-1023). Furthermore, there is concern about the normality and viability of offspring in the following generations after cloning. Here, we report our results related to the latter question and describe the reliability of SCNT to produce healthy GM founder pigs for further studies. From 2006 to 2016, we worked with handmade cloning using donor skin cells from 4 breeds (2 minipigs, 2 standard pigs) that were non-GM or GM with 1 out of more than 20 genes. Cells were reconstructed with oocytes from Large White (LW) sows or gilts, and embryos were in vitro cultured for 5 to 6 days before selection for transfer to LW recipient sows or gilts (Callesen et al. 2014 Cell. Reprogram. 16, 407-410). Enough cloned embryos were produced with each type of GM donor cell for transfer to at least 2 recipients. During the first 4 years, the procedure was being established and refined, whereas in the last 7 years, it was used routinely (Table 1). In the latter period, the GM piglets alive after 30 days represented 17 of the 18 transgenes used. The GM piglets alive 30 days after birth were kept and developed as normal pigs. For 4 of the transgenes used, cloned minipigs were bred using standard breeding; in total, 106 piglets from 15 litters were born in the first generation after the cloning, and 138 piglets from 20 litters in the second generation. Both litter sizes and abnormality frequencies were within the expected range of the given breed (Yucatan or Göttingen), also noting that the pigs harbored a transgene and that some inbreeding was unavoidable due to the few founder piglets available. This work demonstrates that use of SCNT is a reliable way to produce GM founder piglets even though cloning does result in great losses during farrowing and the early postnatal period. However, having overcome these critical phases, the piglets seem to show no visible signs of their challenging background. The overall expense is, of course, high for production of each of the GM founder pigs, and this should be taken into consideration when deciding the species to use for creating a given GM animal model for modelling human diseases. Table 1.Results from 2010 to 2016 of using cloning to produce genetically modified (GM) and non-GM piglets

Genetically modified pigs are becoming an invaluable animal model for agricultural, pharmaceutical, and biomedical applications. Unlike traditional transgenesis, which is accomplished by randomly inserting an exogenous transgene cassette into the natural chromosomal context, targeted genome editing confers precisely editing (e.g., mutations or indels) or insertion of a functional transgenic cassette to user-designed loci. Techniques for targeted genome engineering are growing dramatically and include, e.g., zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems. These systems provide enormous potential applications. In this chapter, we review the use of TALENs for targeted genome editing with focus on their application in pigs. In addition, a brief protocol, including construction of sequence-specific TALENs, delivery of TALENs into primary porcine fibroblasts, and detection of TALEN-mediated cleavage, is described. This chapter is useful for scientists who are inexperienced with TALEN engineering of porcine cells as well as of other large animals.

Chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides have been developed for site-specific correction of episomal and chromosomal target genes. The gene repair approach relies on specific hybridization of the oligonucleotides to the target gene generating a mismatch with the targeted point mutation. Restored gene function is anticipated to occur through activation of endogenous repair systems that recognize the created mismatch. We present an overview of the gene correction results obtained in several target genes by employing various oligonucleotide designs and a discussion of the possible mechanisms underlying the gene correction techniques. Experimental data suggest that modified single-stranded oligonucleotides form intermediate three-stranded heteroduplexes involving the human RecA homologue, hRad51, whereas chimeric RNA/DNA oligonucleotides may participate in three or four-stranded intermediate structures. Protein factors such as hRad52, hRad54, hRPA, and p53 may modulate the heteroduplex formation and participate in the activation of the endogenous mismatch repair and/or nucleotide excision repair pathway(s). The efficiency of the gene correction process may furthermore be influenced by the differential recognition of mismatches by repair enzymes and possible sequence context effects.

Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1(+/∆11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/∆11) fibroblasts. However, all BRCA1(+/∆11) piglets died within 18 days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant inherited skin diseases caused by mutations in either the keratin 5 (K5) or the keratin 14 (K14) genes and characterized by development of intraepidermal skin blisters. The three major subtypes of EBS are Weber-Cockayne, Koebner, and Dowling-Meara, of which the Dowling-Meara form is the most severe. We have investigated five large Danish families with EBS and two sporadic patients with the Dowling-Meara form of EBS. In the sporadic Dowling-Meara EBS patients, a novel K14 mutation (N123S) and a previously published K5 mutation (N176S) were identified, respectively. A novel K14 mutation (K116N) was found in three seemingly unrelated families, whereas another family harbored a different novel K14 mutation (L143P). The last family harbored a novel K5 mutation (L325P). The identified mutations were not present in more than 100 normal chromosomes. Six polymorphisms were identified in the K14 gene and their frequencies were determined in normal controls. These polymorphisms were used to show that the K14 K116N mutation was located in chromosomes with the same haplotype in all three families, suggesting a common ancestor. We observed a strict genotype-phenotype correlation in the investigated patients as the same mutation always resulted in a similar phenotype in all individuals with the mutation, but our results also show that it is not possible to predict the EBS phenotype merely by the location (i.e., head, rod, or linker domains) of a mutation. The nature of the amino acid substitution must also be taken into account.

The gene encoding murine tetranectin (Tna) and its 5'-flanking region was isolated and cloned from a EMBL3 SP6/T7 genomic library. The compiled nucleotide sequence was determined by sequencing, revealing a conserved Tna structure in man and mouse. Mapping of the transcription start point (tsp) suggests that murine Tna has more than one of these. In addition, no consensus TATA-box was found uptream for the putative tsp(s) in the 5'-flanking region of the gene, indicating that the murine Tna promoter belongs to the TATA-less class of genes. The cloned murine Tna was mapped to region F1-F3 on mouse chromosome 9 by fluorescence in situ hybridization (FISH).

Targeted gene editing mediated by chimeric RNA-DNA oligonucleotides (RDOs) or single-stranded oligo-deoxyribonucleotides (ssODNs) has been demonstrated in a wide variety of cell types both in vitro and in vivo. In this study we investigated the correlation between the polarity of the used oligonucleotides and the obtained correction frequency in targeted ssODN-mediated correction of two G>A mutations (introduced at positions 659 and 1567, respectively) in an episomal beta-galactosidase gene. At position 659 the highest correction efficiency was observed using an ssODN complementary to the transcribed strand of the target gene. In contrast, at position 1567 the highest correction frequency was observed using an ssODN complementary to the nontranscribed strand of the target gene. It has been reported that site-specific gene editing mediated by ssODNs targeting the nontranscribed strand of the target gene results in a higher gene editing frequency, and it has been suggested that steric hindrance or displacement of ssODNs by traversing transcription complexes prevents efficient targeting of the transcribed strand. However, the results of the present study demonstrate that occupancy by transcriptional complexes alone does not dictate strand bias in ssODN-mediated gene editing, and that the sequences surrounding the targeted nucleotide may profoundly influence strand bias. This finding has important implications for the design of optimal ssODNs for targeted editing of a given nucleotide sequence.

A full-length cDNA encoding murine tetranectin (TN) was isolated and cloned from a murine lung lambda ZAPII cDNA library. The complete nucleotide sequence was determined revealing an open reading frame encoding 202 amino acids (aa) including a signal peptide of 21 aa. An overall aa identity of 79% exists between the deduced aa sequences of human and murine TN, revealing a high evolutionary conservation of the protein. The highest expression of mouse TN was found in lung and skeletal muscle.

Histidine-rich glycoprotein (HRG) was purified from bovine plasma and the disulphide bridge arrangement established. Disulphide-bridged peptides were obtained from peptic and tryptic degradation of native bovine HRG. Twelve half-cystine residues were found in bovine HRG (compared to sixteen cysteines in human HRG), all involved in the formation of six disulphide bridges connecting Cys-1 to Cys-12, Cys-2 to Cys-3, Cys-4 to Cys-5, Cys-6 to Cys-11, Cys-7 to Cys-8, and Cys-9 to Cys-10. Additional sequence analysis of 14C-carboxymethylated chymotryptic and Staphylococcus aureus V8 protease generated peptides and CNBr-fragments of bovine HRG yielded a partial amino acid sequence of bovine HRG constituting 78% of the sequence when compared to the human cDNA sequence.

Transfer of full-length genes including regulatory elements has been the preferred gene therapy strategy for clinical applications. However, with significant drawbacks emerging, targeted gene alteration (TGA) has recently become a promising alternative to this method. By means of TGA, endogenous DNA repair pathways of the cell are activated leading to specific genetic correction of single-base mutations in the genome. This strategy can be implemented using single-stranded oligodeoxyribonucleotides (ssODNs), small DNA fragments (SDFs), triplex-forming oligonucleotides (TFOs), adeno-associated virus vectors (AAVs) and zinc-finger nucleases (ZFNs). Despite difficulties in the use of TGA, including lack of knowledge on the repair mechanisms stimulated by the individual methods, the field holds great promise for the future. The objective of this review is to summarize and evaluate the different methods that exist within this particular area of human gene therapy research.