Genotype-Independent Transmission of Transgenic Fluorophore Protein by Boar Spermatozoa

Garrels, Wiebke; Holler, Stephanie; Taylor, Ulrike; Herrmann, Doris; Struckmann, C.; Klein, Sabine; Barg-Kues, Brigitte; Nowak‐Imialek, Monika; Ehling, Christine; Rath, Detlef; Ivics, Zoltán; Niemann, Heiner; Kues, Wilfried August

doi:10.1371/journal.pone.0027563

Cited by 16 publications

(23 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Transposase-catalyzed DNA integration seems to favor transcriptionally permissive loci, and all transgenic pigs we have examined, from the founder to the F2 generation showed the expected expression patterns 42,48 . Thus, transposition-mediated transgenesis compares favorably to current alternatives, particularly if the overall success rates of 6-8 % (ratio of transgenic animals with desired phenotype per treated zygotes) and the success rates of 40-60 % of transgenic founders per born animals are taken into account.…”

Section: Transgenesis Using the Sleeping Beauty Transposonmentioning

confidence: 87%

“…One useful way to detect transgene integration and expression is to employ a fluorescent reporter protein, such as Venus, which is an optimized (brighter) derivative of the enhanced yellow fluorescent protein (EYFP). We have previously demonstrated that an SB transposon carrying a CAGGS (CMV enhancer, chicken beta actin promoter)-driven Venus transgene (pT2-RMCE-Venus) facilitates the detection of transgenic animals 38,39,48 .…”

Section: Experimental Designmentioning

confidence: 99%

See 1 more Smart Citation

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

Self Cite

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: 87%

Section: Experimental Designmentioning

confidence: 99%

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, the generation of transgenic pigs with systemic expression of vital fluorophore markers has found widespread interest (311), e.g., for improving gene transfer technologies in pigs (3,5,79,11), for transplantation studies (4,10), for testing the functionality of multicistronic transgene constructs (2), and for visualizing epigenetic phenomena during spermatogenesis and fertilization (6). …”

mentioning

confidence: 99%

“…A litter of 8 cloned piglets was genotyped by standard blotting procedures. According to the restriction of genomic DNA, an internal fragment of 1.4 kb (arrow) and an external fragment (asterisk) of > 3 kb are predictive for hemizygous cloned animals carrying a monomeric integration [for details see (6)]. 1, carrier sow (non-transgenic); 29, cloned piglets; 10, empty; 11, non-related wildtype animal; 12, empty; M, positive control (with bands of 1.4 and 4.9 kb).…”

mentioning

confidence: 99%

Rapid Non-Invasive Genotyping of Reporter Transgenic Mammals

et al. 2012

Self Cite

View full text Add to dashboard Cite

Here we describe a non-invasive method for rapid and highly reproducible genotyping of transgenic mammals with ubiquitous expression of fluorophore reporters. Hair samples from transgenic mice and pigs with systemic expression of the fluorophore reporter Venus were analyzed with a fluorescence microscope in few minutes. The hair samples can be preserved for long-term storage at ambient temperature conditions. This non-invasive method is useful for genotyping of transgenic large animals and contributes to animal welfare by reducing stress and discomfort of the animals during sample collection.

show abstract

“…So far only few studies assessed the toxicity of NP on spermatozoa of farm animals. The recent development of transgenic pigs with fluorophore-loaded spermatozoa (Garrels et al 2011) may be instrumental for establishment of direct comparative tests.…”

Section: R18 D Rath and Othersmentioning

confidence: 99%

Sex selection of sperm in farm animals: status report and developmental prospects

Rath¹,

Barcikowski²,

Graaf³

et al. 2013

Reproduction

View full text Add to dashboard Cite

Pre-selection of spermatozoa based on the relative DNA difference between X-and Y-chromosome bearing populations by flow cytometry is an established method that has been introduced into commercial cattle production. Although several important improvements have increased the sort efficiency, the fertilising ability of sexed spermatozoa based on offspring per insemination is still behind farmers' expectations. The main stress factors, especially on mitochondria, that reduce the lifespan of spermatozoa are described, and new technical as well as biological solutions to maintain the natural sperm integrity and to increase the sorting efficiency are discussed. Among these methods are the identification of Y-chromosome bearing spermatozoa by bi-functionalised gold nanoparticles and triplex hybridisation in vivo as well as new laser-controlled deflection system that replaces the deflection of spermatozoa in the electrostatic field. Additionally, as well as a new nonsurgical transfer system of spermatozoa into the oviduct of cows has been developed and allows a significant reduction of spermatozoa per transfer. Altogether, the improvements made in the recent years will allow a broader use of sex-sorted spermatozoa even in those species that require more cells than cows and sheep.Reproduction (2013) 145 R15-R30

show abstract

Genotype-Independent Transmission of Transgenic Fluorophore Protein by Boar Spermatozoa

Cited by 16 publications

References 62 publications

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

Rapid Non-Invasive Genotyping of Reporter Transgenic Mammals

Sex selection of sperm in farm animals: status report and developmental prospects

Contact Info

Product

Resources

About