Applied Sciences

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14 pages, 6626 KiB

Open AccessFeature PaperArticle

The Creation of a Multiallele Knockout Genotype in Rabbit Using CRISPR/Cas9 and Its Application in Translational Medicine

by Tímea Pintér, Miklós Geiszt, Gábor L. Petheő, Máté Mihálffy, Gabriella Skoda, Nándor Lipták, Andrea Kerekes, Zsuzsanna Bősze, László Hiripi and Lilla Bodrogi

Appl. Sci. 2020, 10(23), 8508; https://doi.org/10.3390/app10238508 - 28 Nov 2020

Viewed by 2806

Abstract

Nonrodent animal models have recently become more valuable in preclinical studies. The limitation of nonrodent animal models is that they must demonstrate relatively reliable and predictable responses in addition to representing complex etiologies of a genetically diverse patient population. In our study, we [...] Read more.

Nonrodent animal models have recently become more valuable in preclinical studies. The limitation of nonrodent animal models is that they must demonstrate relatively reliable and predictable responses in addition to representing complex etiologies of a genetically diverse patient population. In our study, we applied CRISPR/Cas9 technology to produce transgenic rabbits. This approach can be useful for creating genetically divergent and homogeneous populations for studies in translational medicine. NADPH oxidase 4 (NOX4) is a promising therapeutic target, as it is linked to several pathologies including stroke, atherosclerosis, and lung and kidney fibrosis. NOX4 knockout (KO) rabbit lines were created in order to study the in vivo effects resulting from a lack of NOX4 protein and loss of gene function. One of the knockout founders was a germline multiallelic knockout male. Its offspring segregated into three distinct NOX4 knockout and a wild-type lines. Mosaicism is a relatively frequent phenomenon in rabbit transgenesis. Our results point to the possible application of mosaicism in preclinical studies. However, careful planning and evaluation of results are necessary. The predicted off-target sites were studied as well, and no signs of off-target events were detected. Full article

(This article belongs to the Special Issue Rabbit Models for Translational Medicine)

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14 pages, 3158 KiB

Open AccessArticle

Rabbit as an Aging Model in Reproduction: Advanced Maternal Age Alters GLO1 Expression in the Endometrium at the Time of Implantation

by Johanna de Nivelle, Juliane Thoma, Alicia Toto Nienguesso, Tom Seeling, Juliane-Susanne Jung, Anne Navarrete Santos and Maria Schindler

Appl. Sci. 2020, 10(21), 7732; https://doi.org/10.3390/app10217732 - 31 Oct 2020

Cited by 3 | Viewed by 2495

Abstract

Advanced maternal age is associated with adverse pregnancy outcomes and the decline of female fertility in mammals. A potential reason for reduced fertility is metabolic changes due to protein modifications by advanced glycation end products. To elucidate the aging process in female reproduction, [...] Read more.

Advanced maternal age is associated with adverse pregnancy outcomes and the decline of female fertility in mammals. A potential reason for reduced fertility is metabolic changes due to protein modifications by advanced glycation end products. To elucidate the aging process in female reproduction, we analysed a key enzyme for detoxification of reactive dicarbonyls, the glyoxalase 1 (GLO1), in reproductive organs and blastocysts of young and old rabbits at the preimplantation stage. At day 6 post coitum, uterine, oviductal, ovarian tissue and blastocysts from young (16–20 weeks) and old rabbits (>108 weeks) were characterised for GLO1 expression. GLO1 amounts, enzymatic activity and localisation were quantified by qPCR, Simple Western, activity assay and immunohistochemistry. The GLO1 enzyme was present and active in all reproductive tract organs in a cell-type-specific pattern. Ovarian follicle and uterine epithelial cells expressed GLO1 to a high extent. In tertiary follicles, GLO1 expression increased, whereas it decreased in the endometrium of old rabbits at day 6 of pregnancy. In blastocysts of old animals, GLO1 expression remained unchanged. In early pregnancy, advanced maternal age leads to modified GLO1 expression in ovarian follicles and the endometrium, indicating an altered metabolic stress response at the preimplantation stage in older females. Full article

(This article belongs to the Special Issue Rabbit Models for Translational Medicine)

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Review

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21 pages, 624 KiB

Open AccessReview

Rabbit Genetic Resources Can Provide Several Animal Models to Explain at the Genetic Level the Diversity of Morphological and Physiological Relevant Traits

by Luca Fontanesi

Appl. Sci. 2021, 11(1), 373; https://doi.org/10.3390/app11010373 - 2 Jan 2021

Cited by 9 | Viewed by 7055

Abstract

The rabbit (Oryctolagus cuniculus) is a unique multipurpose domestic species that has relevant economic impacts in several contexts. This review is focused on rabbit genetic resources that have been mainly bred for the fixation of differentiating features (e.g., exterior traits) that [...] Read more.

The rabbit (Oryctolagus cuniculus) is a unique multipurpose domestic species that has relevant economic impacts in several contexts. This review is focused on rabbit genetic resources that have been mainly bred for the fixation of differentiating features (e.g., exterior traits) that have been already genetically characterized. Several naturally occurring rabbit mutants could be useful as animal models for the investigation of the biological mechanisms determining their characterizing aspects, with translational potentials. A historical overview of the origin of the domesticated rabbit populations and of their genetic differentiation into many breeds is summarized. Then, a detailed analysis of the genetic features that characterize the different breeds is reported, starting from coat color and coat structure affecting genes (MC1R, ASIP, TYR, MLPH, TYRP1, KIT, LIPH, and FGF5), determining major loci described by classical genetic studies. Mutations in these genes have implications in pigmentation features, hair growth, and related defects. Other gene mutations affecting body size or shapes (HMGA2) and other physiological disfunctions (RORB and BCO2) are also described Additional studies are needed to complete the genetic characterization of some of these loci and to investigate the large genetic variability available in fancy breeds and commercial meat and fur lines. Full article

(This article belongs to the Special Issue Rabbit Models for Translational Medicine)

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18 pages, 2699 KiB

Open AccessReview

Pluripotent Stem Cells for Transgenesis in the Rabbit: A Utopia?

by Worawalan Samruan, Nathalie Beaujean and Marielle Afanassieff

Appl. Sci. 2020, 10(24), 8861; https://doi.org/10.3390/app10248861 - 11 Dec 2020

Viewed by 2757

Abstract

Pluripotent stem cells (PSCs) possess the following two main properties: self-renewal and pluripotency. Self-renewal is defined as the ability to proliferate in an undifferentiated state and pluripotency as the capacity to differentiate into cells of the three germ layers, i.e., ectoderm, mesoderm, and [...] Read more.

Pluripotent stem cells (PSCs) possess the following two main properties: self-renewal and pluripotency. Self-renewal is defined as the ability to proliferate in an undifferentiated state and pluripotency as the capacity to differentiate into cells of the three germ layers, i.e., ectoderm, mesoderm, and endoderm. PSCs are derived from early embryos as embryonic stem cells (ESCs) or are produced by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). In mice, PSCs can be stabilized into two states of pluripotency, namely naive and primed. Naive and primed PSCs notably differ by their ability to colonize a host blastocyst to produce germline-competent chimeras; hence, naive PSCs are valuable for transgenesis, whereas primed PSCs are not. Thanks to its physiological and developmental peculiarities similar to those of primates, the rabbit is an interesting animal model for studying human diseases and early embryonic development. Both ESCs and iPSCs have been described in rabbits. They self-renew in the primed state of pluripotency and, therefore, cannot be used for transgenesis. This review presents the available data on the pluripotent state and the chimeric ability of these rabbit PSCs. It also examines the potential barriers that compromise their intended use as producers of germline-competent chimeras and proposes possible alternatives to exploit them for transgenesis. Full article

(This article belongs to the Special Issue Rabbit Models for Translational Medicine)

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19 pages, 641 KiB

Open AccessReview

Hyperlipidemic Rabbit Models for Anti-Atherosclerotic Drug Development

by Manabu Niimi, Yajie Chen, Haizhao Yan, Yao Wang, Tomonari Koike and Jianglin Fan

Appl. Sci. 2020, 10(23), 8681; https://doi.org/10.3390/app10238681 - 4 Dec 2020

Cited by 6 | Viewed by 3822

Abstract

Hyperlipidemia or dyslipidemia is a major risk factor for atherosclerotic diseases. Experimental animals play an important role in elucidating the molecular mechanisms of the pathophysiology of hyperlipidemia as well as in drug development. Rabbits are one of the most suitable models to study [...] Read more.

Hyperlipidemia or dyslipidemia is a major risk factor for atherosclerotic diseases. Experimental animals play an important role in elucidating the molecular mechanisms of the pathophysiology of hyperlipidemia as well as in drug development. Rabbits are one of the most suitable models to study human hyperlipidemia because many features of the lipoprotein metabolism of rabbits are similar to those of humans such as LDL-rich lipoproteins in plasma, apolipoprotein B mRNA editing, and cholesteryl ester transfer protein. Currently, three types of rabbit models are commonly used for studying hyperlipidemia: (1) diet-induced hyperlipidemic rabbits, (2) spontaneous hyperlipidemic rabbits, and (3) gene-manipulated rabbits (transgenic and knockout rabbits). In this review, we give an overview of the features of hyperlipidemic rabbits and discuss the usefulness of rabbits for the development of anti-atherogenic drugs. Full article

(This article belongs to the Special Issue Rabbit Models for Translational Medicine)

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15 pages, 324 KiB

Open AccessReview

Transgenic Rabbit Models: Now and the Future

by Fumikazu Matsuhisa, Shuji Kitajima, Kazutoshi Nishijima, Toshiaki Akiyoshi, Masatoshi Morimoto and Jianglin Fan

Appl. Sci. 2020, 10(21), 7416; https://doi.org/10.3390/app10217416 - 22 Oct 2020

Cited by 14 | Viewed by 6485

Abstract

Transgenic rabbits have contributed to the progress of biomedical science as human disease models because of their unique features, such as the lipid metabolism system similar to humans and medium body size that facilitates handling and experimental manipulation. In fact, many useful transgenic [...] Read more.

Transgenic rabbits have contributed to the progress of biomedical science as human disease models because of their unique features, such as the lipid metabolism system similar to humans and medium body size that facilitates handling and experimental manipulation. In fact, many useful transgenic rabbits have been generated and used in research fields such as lipid metabolism and atherosclerosis, cardiac failure, immunology, and oncogenesis. However, there have been long-term problems, namely that the transgenic efficiency when using pronuclear microinjection is low compared with transgenic mice and production of knockout rabbits is impossible owing to the lack of embryonic stem cells for gene targeting in rabbits. Despite these limitations, the emergence of novel genome editing technology has changed the production of genetically modified animals including the rabbit. We are finally able to produce both transgenic and knockout rabbit models to analyze gain- and loss-of-functions of specific genes. It is expected that the use of genetically modified rabbits will extend to various research fields. In this review, we describe the unique features of rabbits as laboratory animals, the current status of their development and use, and future perspectives of transgenic rabbit models for human diseases. Full article

(This article belongs to the Special Issue Rabbit Models for Translational Medicine)

15 pages, 1094 KiB

Open AccessReview

Immunodeficient Rabbit Models: History, Current Status and Future Perspectives

by Jun Song, Brooke Pallas, Dongshan Yang, Jifeng Zhang, Yash Agarwal, Y. Eugene Chen, Moses Bility and Jie Xu

Appl. Sci. 2020, 10(20), 7369; https://doi.org/10.3390/app10207369 - 21 Oct 2020

Cited by 2 | Viewed by 6648

Abstract

Production of immunodeficient (ID) models in non-murine animal species had been extremely challenging until the advent of gene-editing tools: first zinc finger nuclease (ZFN), then transcription activator-like effector nuclease (TALEN), and most recently clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR)/Cas9. We [...] Read more.

Production of immunodeficient (ID) models in non-murine animal species had been extremely challenging until the advent of gene-editing tools: first zinc finger nuclease (ZFN), then transcription activator-like effector nuclease (TALEN), and most recently clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR)/Cas9. We and others used those gene-editing tools to develop ID rabbits carrying a loss of function mutation in essential immune genes, such as forkhead box protein N1 (FOXN1), recombination activating gene 1/2 (RAG1/2), and interleukin 2 receptor subunit gamma (IL2RG). Like their mouse counterparts, ID rabbits have profound defects in their immune system and are prone to bacterial and pneumocystis infections without prophylactic antibiotics. In addition to their use as preclinical models for primary immunodeficient diseases, ID rabbits are expected to contribute significantly to regenerative medicine and cancer research, where they serve as recipients for allo- and xeno-grafts, with notable advantages over mouse models, including a longer lifespan and a much larger body size. Here we provide a concise review of the history and current status of the development of ID rabbits, as well as future perspectives of this new member in the animal model family. Full article

(This article belongs to the Special Issue Rabbit Models for Translational Medicine)

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Other

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5 pages, 1377 KiB

Open AccessBrief Report

The Genetic Mutation of ANO5 in Rabbits Recapitulates Human Cardiomyopathy

by Tingting Sui, Hongwu Yao, Tao Zhang, Jinze Li, Liangxue Lai and Zhanjun Li

Appl. Sci. 2020, 10(14), 4976; https://doi.org/10.3390/app10144976 - 20 Jul 2020

Cited by 1 | Viewed by 1934

Abstract

The limb girdle muscular dystrophy type 2L (LGMD2L) is caused by mutations of the ANO5 gene in humans which encodes a 913 amino-acid integral membrane protein. Although cardiomyopathy has been reported in patients with an ANO5 mutation, the ANO5 mutant mice did not [...] Read more.

The limb girdle muscular dystrophy type 2L (LGMD2L) is caused by mutations of the ANO5 gene in humans which encodes a 913 amino-acid integral membrane protein. Although cardiomyopathy has been reported in patients with an ANO5 mutation, the ANO5 mutant mice did not recapitulate this phenotype in previous studies. This study demonstrated that the ANO5^−/− rabbits recapitulated the typical signs of cardiomyopathy with decreased ejection fraction (EF) and fraction shortening (FS) with increased interstitial fibrosis. This ANO5^−/− rabbit model would promote basic research to comprehend the pathogenesis and mechanism of ANO5-related cardiomyopathy. Full article

(This article belongs to the Special Issue Rabbit Models for Translational Medicine)

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