Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression

Yang, Dongshan; Song, Jun; Zhang, Jifeng; Xu, Jie; Zhu, Tianqing; Wang, Zhong; Lai, Liangxue; Chen, Y. Eugene

doi:10.1038/srep25161

Cited by 40 publications

(43 citation statements)

References 25 publications

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“…RAG1/2 and IL2RG mutant rabbits have been reported by Lai’s group previously using TALEN and Cas9 10 , 29 . They also produced DGM rabbits of IL2RG and RAG1 by Cas9.…”

Section: Discussionmentioning

confidence: 85%

Production of immunodeficient rabbits by multiplex embryo transfer and multiplex gene targeting

Song

Yang

Ruan

et al. 2017

Sci Rep

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Immunodeficient mice have been used predominantly in biomedical research. Realizing that large animal species may have an enhanced ability to predict clinical outcome relative to mice, we worked to develop immunodeficient rabbits by CRISPR/Cas9. We first demonstrated that multiplex embryo transfer efficiently produced multiple lines of single-gene mutant (SGM) founders. Embryos microinjected with single sgRNA targeting FOXN1, RAG2, IL2RG or PRKDC were pooled for embryo transfer. As few as three recipients were used to produce twenty SGM founders for four genes. We then demonstrated the powerful multiplex targeting capacity of CRISPR/Cas9. First, two genes on the same chromosome were targeted simultaneously, resulting in three RAG1/RAG2 double-gene mutant (DGM) founders. Next we microinjected forty-five embryos each with five sgRNAs targeting FOXN1, RAG1, RAG2, IL2RG and PRKDC, and transferred them to two recipients. Five founders were produced: one SGM, two DGM, one triple-gene mutant and one quadruple-gene mutant. The present work demonstrates that multiplex embryo transfer and multiplex gene targeting can be used to quickly and efficiently generate mutant rabbit founders. Four lines of SGM (e.g. FOXN1, RAG2, IL2RG, and PRKDC) immunodeficient rabbits, as well as multigenic mutant immunodeficient rabbits have been produced. These animals may prove useful for biomedical research.

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“…RAG1/2 and IL2RG mutant rabbits have been reported by Lai’s group previously using TALEN and Cas9 10 , 29 . They also produced DGM rabbits of IL2RG and RAG1 by Cas9.…”

Section: Discussionmentioning

confidence: 85%

Production of immunodeficient rabbits by multiplex embryo transfer and multiplex gene targeting

Song

Yang

Ruan

et al. 2017

Sci Rep

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“…We used TALEN mRNA microinjection into pronuclear stage rabbit embryos (20) rather than the CRISPR/Cas9 system (40, 43) because we envisaged that for embryo injections the toxicity and off-target effects of the former are easier to control (44), although recent improvements of the CRISPR/Cas9 technique could solve this issue (45). FAH −/− rabbits display liver and kidney manifestations of the human genetic disorder but also have frequent ocular alterations (mostly corneal keratitis).…”

Section: Discussionmentioning

confidence: 99%

Fumarylacetoacetate Hydrolase Knock-out Rabbit Model for Hereditary Tyrosinemia Type 1

Zhang

Yang

et al. 2017

Journal of Biological Chemistry

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Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH−/− rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH−/− rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH−/− rabbits are an attractive alternative for modeling the consequences of HT1.

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“…This is phenomenon is called mosaicism, as it results in mosaic individuals composed by more than one cell population. Mosaicism was initially overlooked, as it is not a common problem in the generation of murine KO models (Bermejo-Alvarez et al, 2015), but most of the publications that have performed allele screening following CRISPR direct injection in zygotes have observed mosaicism in different species such as pigs (Hai et al, 2014;Sato et al, 2015;Wang et al, 2015c;Chuang et al, 2016;Kang et al, 2016;Petersen et al, 2016;Yu et al, 2016;Zhou et al, 2016;Burkard et al, 2017;Park et al, 2017;Whitworth et al, 2017), goats ), sheep (Crispo et al, 2015Wang et al, 2016c;Vilarino et al, 2017;Zhang et al, 2017), cattle (Bevacqua et al, 2016 and rabbits (Yan et al, 2014;Honda et al, 2015;Guo et al, 2016;Lv et al, 2016;Song et al, 2016a;Song et al, 2016b;Sui et al, 2016;Yang et al, 2016;.…”

Section: Mosaicism Impairs Direct Ko Generation By Crisprmentioning

confidence: 99%

Directions and applications of CRISPR technology in livestock research

Lamas-Toranzo¹,

Ramos‐Ibeas²,

Pericuesta³

et al. 2018

Anim Reprod

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The ablation (KO) or targeted insertion (KI) of specific genes or sequences has been essential to test their roles on a particular biological process. Unfortunately, such genome modifications have been largely limited to the mouse model, as the only way to achieve targeted mutagenesis in other mammals required from somatic cell nuclear transfer, a time-and resource-consuming technique. This difficulty has left research in livestock species largely devoided of KO and targeted KI models, crucial tools to uncover the molecular roots of any physiological or pathological process. Luckily, the eruption of site-specific endonucleases, and particularly CRISPR technology, has empowered farm animal scientists to consider projects that could not develop before. In this sense, the availability of genome modification in livestock species is meant to change the way research is performed on many fields, switching from descriptive and correlational approaches to experimental research. In this review we will provide some guidance about how the genome can be edited by CRISPR and the possible strategies to achieve KO or KI, paying special attention to an initially overlooked phenomenon: mosaicism. Mosaicism is produced when the zygote´s genome edition occurs after its DNA has replicated, and is characterized by the presence of more than two alleles in the same individual, an undesirable outcome when attempting direct KO generation. Finally, the possible applications on different fields of livestock research, such as reproduction or infectious diseases are discussed. ReferencesBennett GL, Leymaster KA. 1989. Integration of ovulation rate, potential embryonic viability and uterine capacity into a model of litter size in swine. J Anim Sci, 67:1230-1241. Berg DK, van Leeuwen J, Beaumont S, Berg M, Pfeffer PL. 2010. Embryo loss in cattle between Days 7 and 16 of pregnancy. Theriogenology, 73:250-260. Berg DK, Smith CS, Pearton DJ, Wells DN, Broadhurst R, Donnison M, Pfeffer PL. 2011. Trophectoderm lineage determination in cattle. Dev Cell, 20:244-255. Bermejo-Alvarez P, Rizos D, Rath D, Lonergan P, Gutierrez-Adan A. 2010. Sex determines the expression level of one third of the actively expressed genes in bovine blastocysts. Proc Natl Acad Sci U S A,

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Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression

Cited by 40 publications

References 25 publications

Production of immunodeficient rabbits by multiplex embryo transfer and multiplex gene targeting

Production of immunodeficient rabbits by multiplex embryo transfer and multiplex gene targeting

Fumarylacetoacetate Hydrolase Knock-out Rabbit Model for Hereditary Tyrosinemia Type 1

Directions and applications of CRISPR technology in livestock research

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