Dataset Information

Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon.

ABSTRACT: Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fluorescent protein (eGFP) gene by injecting pronuclei with PB plasmids. We showed that the transgenic rats expressed the RFP or eGFP gene in many organs and had the capability to transmit the marker gene to the next generation through germline integration. In addition, rat embryonic stem cells (ESCs) carrying an RFP reporter gene can be derived from the blastocysts of the transgenic rats. Moreover, the RFP gene can be detected in chimeras derived from RFP ESCs via blastocyst injection. This work suggests that PB-mediated transgenesis is a powerful tool to generate transgenic rats expressing fluorescent proteins with high efficiency, and this technique can be used to derive rat ESCs expressing a reporter protein.

SUBMITTER: Li T

PROVIDER: S-EPMC5021943 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon.

Li Tianda T Shuai Ling L Mao Junjie J Wang Xuepeng X Wang Mei M Zhang Xinxin X Wang Leyun L Li Yanni Y Li Wei W Zhou Qi Q

Scientific reports 20160914

Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fl ...[more]

PMID: 27624004

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Project description:BackgroundLivestock, particularly cattle, are crucial for biotechnology fields, such as genetic breeding, infectious diseases, bioreactors, and specific disease models. However, genetic engineering in cattle has lagged due to long gestation periods, single embryo pregnancies, and high rearing costs. Additionally, the slow validation of germline transmission and the absence of germline-competent embryonic stem cells hinder progress. With the development of genome editing technologies like ZFN, TALEN, and CRISPR-Cas9, recent advancements have shown that Cas9-expressing pigs and chickens have been successfully produced. We hypothesize that generating CRISPR/Cas9-expressing cattle and their resources will provide a powerful resource for bovine genome editing, advancing our understanding of bovine genetics and disease resistance.ResultsIn this study, two types of Cas9-expressing cattle were successfully produced: Cas9-RFP-fatty acid dehydrogenase I (FatI), Cas9-GFP-sgRNA for the prion protein (sgPRNP). Somatic cells from these cattle were induced to mutate multiple target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene (Cas9, reporter genes, FatI), and sgPRNP) to the next generation. Furthermore, the gene editing capabilities of Cas9, including knockout and high-efficiency knock-in, were confirmed in embryos derived from F1 semen through in vitro production.ConclusionThese data demonstrate, for the first time, that Cas9-expressing cattle were successfully born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, somatic and germ cells derived from F0 and F1generations were used to evaluate the potential for gene editing (knockout and knock-in) in multiple genes. PRNP-mutated F1 cattle are currently being raised as a resistance model for bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will serve as a valuable resource for both in vitro and in vivo genome editing, advancing our genetic understanding of bovine genomics and diseases.

Dataset Information

Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon.

Publications

Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon.

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