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Mead Lab: Methods

Xenopus Methods

• Transposon transgenesis

  Recent publications from our laboratory outline the injection-mediated transgenesis method we use:

  Donald A. Yergeau, Emin Kuliyev and Paul E. Mead. (2007) Injection-mediated transposon transgenesis in Xenopus tropicalis and the identification of integration sites by modified extension primer tag selection (EPTS) linker-mediated PCR. Nature Protocols 2(11):2975-2986.

  Donald A. Yergeau, Clair M. Kelley, Haiqing Zhu, Emin Kuliyev and Paul E. Mead. (2010) Transposon transgenesis in Xenopus. Methods 51:92-100.

  Donald A. Yergeau, Michelle R. Johnson Hamlet, Emin Kuliyev, Haiqing Zhu, Joanne R. Doherty, Taylor D. Archer, Andrea P. Subhawong, Marc B. Valentine, Clair M. Kelley and Paul E. Mead. (2009) Transgenesis in Xenopus Using the Sleeping Beauty Transposon System. Developmental Dynamics 238(7):1727-1743. PMCID: PMC2848081.
  
  

• Implanting RFID micro-chips

  Micro-chips and Readers are from Microsensys (www.microsensys.de).

  
  

  The method we use for isoflurane anesthesia and implanting the chips is provide here.

  Donald A. Yergeau, Clair M. Kelley, Haiqing Zhu, Emin Kuliyev and Paul E. Mead. (2010) Transposon transgenesis in Xenopus. Methods 51:92-100.
  
  

• Reading RFID micro-chips

  
  
  

• Sorting Transgenic Frogs

  

  
  
  GFP Goggles
  We use a various methods for identifying transgenic animals in an outcross population. For large tadpoles and adult frogs the "GFP miners lamp" from BLS works well to quickly sort animals.

  Emin wearing the GFP goggles - the goggles work best with the room lights off!

  
  

• Swab Test for Genotyping Adult Frogs (Swamp CSI)

  We use buccal swabs to collect skin samples from the frog - working on the premise that the inside of our mouth is like the outside of a frog. The frog is held in one hand and gently swabbed to collect a few skin cells for genotyping. We swab the exposed areas of the frog skin for ~5-10 seconds on each side of the swab to ensure enough cells are collected for PCR analysis. The frog is isolated in a labeled container until the PCR analysis is complete, usually the next day. We use two commercial systems for swabbing the frog: either the DDK50-SK2 kit from Isohelix; this kit contains 50 swabs and reagents for DNA isolation) or the Copan 4N6 Forensic Collection System (www.copanusa.com; cat # 3520CA).

  

  Copan 4N6 flocked swabIsohelix SK2 buccal swab

  The cells are disrupted with 0.5 ml of buffer containing proteinase K for one hour at 60oC and then the swab is removed from the tube. The DNA is precipitated and resuspended overnight at 4oC in 100 ul of TE. Standard genomic PCR reactions, using 10 ul of the resuspended DNA per reaction, are set up to determine the genotype of the animal. We use primer sets that are specific for the transposon transgene and also include a primer set for an unrelated wild type genomic locus as a positive control for DNA recovery. The final DNA concentration will be low, and up to 40 cycles of PCR may be needed to see the amplified band on a standard ethidium bromide-stained agarose gel. This method is fast and non-invasive, and is useful for a variety of applications. For example, transposon transgenic founder animals may have multiple integration events that independently segregate in the outcross population. The specific alleles inherited by an individual F1 frog can be quickly determined using the skin swab test; and this test can be performed well before the animal has reached maturity. Animals with the desired genotypes can be selected for subsequent outcross. Furthermore, some cell-specific transgenes may be difficult to visualize in the adult frog and the swab test can be used to identify 'positive' adult animals in an outcross population.

Molecular Biology

• EPTS LM-PCR
  

  Extension Primer Tag Selection Linker-Mediated PCR (EPTS LM-PCR) is the method we use to clone the integration sites of transposon transgenes in the frog genome. A detailed method can be found in:

  Donald A. Yergeau, Emin Kuliyev and Paul E. Mead. (2007) Injection-mediated transposon transgenesis in Xenopus tropicalis and the identification of integration sites by modified extension primer tag selection (EPTS) linker-mediated PCR. Nature Protocols 2(11):2975-2986.

  An updated version of this method, including minor modifications due to the current availability of certain commercial reagents, is provided here.

• Genomic DNA from tadpoles
• Southern analysis
• In situ hybridization
• Whole Mount Immunohistochemistry
• β-gal staining
• TUNEL staining