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Mead Lab: Transgenic Frogs

Xenopus laevis

pT2βGFP

• Two lines available with ubiquitous GFP expression driven from a CAGGS synthetic promoter. Sleeping Beauty transposon transgenic .

  Yergeau DA, Johnson Hamlet MR, Kuliyev E, Zhu H, Doherty JR, Archer TD, Subhawong AP, Valentine MB, Kelley CM and Mead PE. (2009) Transgenesis in Xenopus using the Sleeping Beauty transposon system. Dev Dyn. 238(7):1727-43.

pT2flk1GFP

• Vascular-specific GFP expression driven from the Xenopus laevis flk1 promoter and first intron enhancer. Sleeping Beauty transposon transgenic.

  Doherty JR, Johnson Hamlet MR, Kuliyev E and Mead PE. (2007) A flk-1 promoter/enhancer reporter transgenic Xenopus laevis generated using the Sleeping Beauty transposon system: an in vivo model for vascular studies. Dev Dyn. 236(10):2808-17.

Xenopus tropicalis

pT2βGFP

• More than ten lines available with ubiquitous GFP expression driven from a CAGGS synthetic promoter. Sleeping Beauty transposon transgenic.

  Yergeau DA, Johnson Hamlet MR, Kuliyev E, Zhu H, Doherty JR, Archer TD, Subhawong AP, Valentine MB, Kelley CM and Mead PE. (2009) Transgenesis in Xenopus using the Sleeping Beauty transposon system. Dev Dyn. 238(7):1727-43.

Tol2XIG

• Multiple lines available with ubiquitous GFP expression driven from a Xenopus EF1a promoter. Tol2-mediated transgenics. The 279 bp Xenopus EF1a promoter element acts as an efficient enhancer trap.

  Hamlet MR, Yergeau DA, Kuliyev E, Takeda M, Taira M, Kawakami K and Mead PE. (2006) Tol2 transposon-mediated transgenesis in Xenopus tropicalis. Genesis. 44(9):438-45.

  Yergeau DA, Kelley CM, Kuliyev E, Zhu H, Sater AK, Wells DE and Mead PE. (2010) Remobilization of Tol2 transposons in Xenopus tropicalis. BMC Dev Biol. 10:11.

Tol2XIG 10M

• The 10M line has a single-copy transposon insertion on scaffold 246 at base position 110097 (246:110079). Scaffold 246 maps to Linkage Group 5 = chromosome 8. The chromosomal location has been verified by FISH analysis with a Tol2XIG probe. The integration site lies within an intron of the D4 gene. Incrosses of this line give viable, fertile homozygous adults.

F1 10M tadpoles

Integration site for the Tol2XIG 10M line (246:110079) is in an intron of the D4 gene.

FISH analysis of metaphase chromosomes from the Tol2XIG 10M line.

PCR genotyping the 10M locus.

Tol2XIG 12M

• The founder of the 12M line has four integration events that can be segregated by outcross. The four alleles were called Soul Patch (slp, due to the intense GFP expression in the basibranchial-basihyal (BB) cartilage resembling the facial hair worn below the lower lip), Handlebar (hbr, in keeping with the facial hair theme inspired by slp, we named the other alleles in the 12M family after facial hair styles), Garibaldi (grb) and Chinstrap (chs).

The four alleles from founder Tol2XIG 12M.

Integration site analysis for the 12M alleles. Target site duplication is underlined. Tol2 sequence is in lowercase. Genomic sequence is in uppercase. *The slp integration is in a repeat and could reside on one of several scaffolds - we have provisionally identified scaffold 42 as the most likely integration site.

The Tol2XIG 12M^hrb line was used for a substrate in an injection-mediated remobilization strategy where transgenic embryos were injected at the one-cell stage with mRNA encoding the Tol2 transposase enzyme. The injected embryos were raised and outcrossed and the progeny were analyzed for novel re-integration events. We have identified four novel alleles from a single 12M^hrb remobilized frog and each allele has a distinct GFP expression pattern.

Tol2XIG 12M hbr remobilized alleles

• The remobilization events from 12M^hbr were called jovan heat (joh), centaure (cen), folique (fol) and brut (bru). The donor alleles were named after facial hair styles; the remobilized alleles were given names of shaving products.

Integration sites for the parental allele hbr and the four remobilized alleles found in the progeny of the Tol2 mRNA injected 12M^hbr donor. Target site duplication is underlined. Tol2 sequence is in lowercase. Genomic sequence is in uppercase.

Examples of the GFP expression patterns of three remobilized alleles compared to the parental 12M^hbr donor.

Chromosome map of the remobilization events from the 12M^hbr donor site (thick line labeled hbr). The fol re-integration event is a local hop. The cen, bru and joh alleles are on separate chromosomes and can be segregated by simple outcross.

The jovan heat (joh) allele integrated into a HEAT repeat gene, in an intron near the 3' end of the gene. The joh allele has robust expression of GFP in the pronephros.

GFP expression patterns of the (a) hbr and (b) joh integration events. joh has intense GFP expression in the developing kidney (arrows). (c) Close up view of fluorescence in the right kidney. Arrowheads point to the eye for orientation. Adapted from Yergeau DA, Kelley CM, Zhu H, Kuliyev E and Mead PE. (2010) Transposon transgenesis in Xenopus. Methods 51:92-100.

The Tol2XIG insertion site of the joh allele is ~46 kB away from the first exon of the HNF1ß, a gene expressed at high levels in the developing kidney and the joh allele may represent an enhancer trap of HNF1ß.

The joh allele is ~46 kb upstream of the HNF1b gene (a) which is expressed in the developing kidney (b & c).  Figure is adapted from Yergeau DA, Kelley CM, Kuliyev E, Zhu H, Sater AK, Wells DE and Mead PE. (2010) Remobilization of Tol2 transposons in Xenopus tropicalis.  BMC Dev Biol. 10:11.

Last Updated 5/12/10