De novo methylation and expression of retroviral genomes during mouse embryogenesis

Jähner, Detlev; Stuhlmann, Heidi; Stewart, Colin L.; Harbers, Klaus; Löhler, Jürgen; Simon, Iva; Jaenisch, Rudolf

doi:10.1038/298623a0

Cited by 492 publications

(264 citation statements)

References 43 publications

Supporting

Mentioning

251

Contrasting

Order By: Relevance

“…Furthermore, unlike retroviral vectors [43][44][45][46] , SB100X transposase-catalyzed transgene integration does not seem to trigger transcriptional silencing 34,39 . Importantly, SB transposon-tagged genomic loci are suitable for recombinase-mediated cassette exchange (RMCE) 47 , allowing targeted genome engineering 5,42 .…”

Section: Transgenesis Using the Sleeping Beauty Transposonmentioning

confidence: 98%

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

View full text Add to dashboard Cite

2The pig has emerged as an important large animal model in biomedical and pharmaceutical research.We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer, and offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing as well as the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in one day; generation of germline-transgenic lines by using this protocol takes approximately one year.

show abstract

Section: Transgenesis Using the Sleeping Beauty Transposonmentioning

confidence: 98%

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

View full text Add to dashboard Cite

show abstract

“…[11][12][13][14] The retroviral vector driving the stable gene expression in the mouse brain is required in the adult brain of the organism to be studied. In primary neural stem cells, the gene expression from the CE vector containing the cellular promoter was shut down more easily than those from MT, MS and MP vectors containing viral LTRs.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, when the same titer of the CE and MS vectors was coinjected into the embryonic brain, the MS vector drove the expression of the transgenes more stably than the CE vector in the adult mouse brain. It has previously been thought that the main reason for the retroviral silencing was the de novo methylation of CpG sequence in viral promoters, 11,12,14 and thus the use of a cellular promoter as an internal promoter had been expected to confer resistance to silencing. However, this does not seem to be the case for the CE vector.…”

Section: Discussionmentioning

confidence: 99%

A retroviral vector suitable for ultrasound image-guided gene delivery to mouse brain

et al. 2011

View full text Add to dashboard Cite

Gene transfer to the early-stage embryonic brain using the ultrasound image-guided gene delivery (UIGD) technique has proven to be valuable for investigating brain development. Thus far, this technology has been restricted to the study of embryonic neurogenesis. When this technique is designed to be employed for the study in adult animals, a long-term stable gene expression will be required. We attempted to develop a retroviral vector suitable for expressing exogenous genes in the brains of postnatal and adult mice in the context of the UIGD technique. Retroviral vectors containing four different long terminal repeats (LTRs) (each from Moloney murine leukemia virus (MoMLV), murine stem cell virus (MSCV), myeloproliferative sarcoma virus (MPSV) and spleen focus-forming virus (SFFV)) were compared using the well-known CE vector having the EF1a internal promoter as a control. The MS vector containing MSCV LTR produced a higher viral titer and a higher level of gene expression than other vectors including CE. The MS vector drove the gene expression in cultured neural stem cells for 3 weeks. Furthermore, the MS vector could efficiently deliver the gene to the mouse central nervous system, as transgene expression was found in various regions of the brains and spinal cords as well as in all major neural cell types. The data from an in vivo luciferase imaging analysis showed that the gene expression from the MS vector was sustainable for almost 3 months. Our data suggested that the MS vector would be suitable to construct mice containing the transgene expressed in the brain or spinal cord in a quick and cost-effective manner.

show abstract

“…Furthermore, unlike retroviral vectors [34][35][36][37] , SB100X transposase-catalyzed transgene integration does not seem to trigger transcriptional silencing 24,30 . Therefore, the application of the Sleeping Beauty transposon system described here can significantly enhance the rabbit genomic toolbox.…”

Section: Transgenesis With the Sleeping Beauty Transposonmentioning

confidence: 98%

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

et al. 2014

View full text Add to dashboard Cite

2The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for highefficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNA encoding the SB100X hyperactive transposase, together with plasmid DNA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes approximately two months.Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection, and offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) to lentiviral approaches, without limitations on vector design, issues of transgene silencing as well as the toxicity and biosafety concerns of working with viral vectors.

show abstract

De novo methylation and expression of retroviral genomes during mouse embryogenesis

Cited by 492 publications

References 43 publications

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

A retroviral vector suitable for ultrasound image-guided gene delivery to mouse brain

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

Contact Info

Product

Resources

About