Rapid Non-Invasive Genotyping of Reporter Transgenic Mammals

Garrels, Wiebke; Cleve, Nicole; Niemann, Heiner; Kues, Wilfried A.

doi:10.2144/000113874

Cited by 8 publications

(14 citation statements)

References 20 publications

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“…This gene dose effect has been noticed previously in newborn pups (Cao et al, 2005;Zhu et al, 2005) and adult mice (Cornett et al, 2011;Garrels et al, 2012). However, a reliable method for objective quantification of fluorescence protein expression has not yet been provided.…”

Section: Introductionsupporting

confidence: 58%

“…Distinct fluorescence levels of different genotypes have been noticed before in newborn and adult transgenic mice ubiquitously expressing eGFP, Venus and mRFP1 (Cao et al, 2005;Garrels et al, 2012;Zhu et al, 2005). However, positioning and immobilization of newborn pups within an in vivo imaging device remains a challenge for accurate measurements.…”

Section: Discussionmentioning

confidence: 99%

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Non-invasive instant genotyping of fluorescently labeled transgenic mice

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Yau

Kolbe

et al. 2015

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SummaryFluorescence proteins have been useful as genetic reporters for a wide range of applications in biomedical research and are frequently used for the analysis of transgene activity. Here, we show that expression levels of the ubiquitously expressed fluorescent proteins eGFP, mCherry and tdTomato can be measured in transgenic mouse lines with random or targeted integrations. We identified the tail of the mouse as the tissue best suited for quantifying fluorescence intensity and show that expression levels in the tail correlate with gene dose. This allows for instant non-invasive determination of the genetic condition at the transgenic locus (hemizygous/ heterozygous and homozygous) while simultaneously providing an objective comparison for transgene expression levels among different mouse lines. In summary, we demonstrate for the first time that the gene dose of a ubiquitously expressed fluorescence reporter can be reliably quantified and directly linked to the genotype of transgenic mice. Based on this information, animals with the appropriate genotype can be instantly selected without laborious analysis for establishing and breeding of new transgenic lines, reducing the number of "waste" animals. Furthermore, no tissue sampling is necessary, which is a significant refinement of genotyping procedures. Both aspects are important improvements for the genotyping of transgenic mice that follow the principles of the 3Rs (reduction and refinement).

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Section: Introductionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Non-invasive instant genotyping of fluorescently labeled transgenic mice

Fink

Yau

Kolbe

et al. 2015

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“…The expression pattern can be confirmed by Western blotting with a transgene-specific antibody (Fig. 7e, f) 59 . Primer SB short (10 pmol/μl) ( Table 1) Primer Tbal rev (10 pmol/μl) ( Table 1 Supplementary Fig.…”

Section: Critical To Avoid False Positive Detections and To Set Up Thmentioning

confidence: 79%

“…Images and videos can be obtained by using an electronic camera equipped with the proper emission filter (Supplementary Video 1). 79 (Optional) Hair phenotyping: collect hair samples for fluorescence analysis under a microscope 59 . In transgenic pigs carrying the CAGGS-Venus transposon, the Venus protein is deposited in the hair and maintains its fluorescent properties.…”

Section: Critical To Avoid False Positive Detections and To Set Up Thmentioning

confidence: 99%

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Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

et al. 2014

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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.

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