Welcome to Gaia! :: View User's Journal | Gaia Journals

Genotyping. Yolk sacs from 10.5-dpc embryos or toe clips from 2-week-old pups were used to extract DNA for PCR genotyping. The presence of the Gtrosa26tm1Sor allele and the excised Gtrosa26tm1Sor allele (named Gtrosa26lacZ) was determined by PCR using three primers together: F1295, 5′-ggttgaggacaaactcttcgc-3′; Pgk-As, 5′-ctgactaggggaggagtagaag-3′; lacZ-As, 5′-gctgcaaggcgattaagttgg-3′. The F1295 and Pgk-As primers amplify VX950 337-bp product specific for the Gtrosa26tm1Sor allele, and primers F1295 and lacZ-As amplify a 255-bp product specific for the Gtrosa26lacZ allele. PCR products were resolved and visualized by 1.2% agarose gel electrophoresis.
Results
Purification of HTNCre fusion protein and HTNCre-mediated excision in vitro
Fig. 1.
Purification of Cre fusion protein and analysis of Cre activity in vitro. (A) Modular structure of recombinant Cre. H, 6xHis; T, a sex hormones protein transduction peptide; N, a nuclear localization sequence. (B) SDS–PAGE analysis of purified HTNCre. M, Kaleidoscope molecular weight marker (Bio-Rad); CL, cleared lysate; FT, flow through; W1, wash fraction 1; W2, wash fraction 2; E, eluted fraction; C, Microcon concentrate. The arrow indicates purified Cre. (C) The diagram shows Cre-mediated excision where Cre induces excision reaction between two loxP (arrows), one in the linear plasmid and the other in the annealed loxP. (D) Cre activity was analyzed by incubation at 37 °C for 20 min with indicated concentrations of HTNCre (left); Cre activity over time was analyzed by incubating the substrates with 0.5 μM of HTNCre at 37 °C for the indicated times (right). The cleaved DNA products were separated by agarose gel electrophoresis. M, 1 kb Plus DNA marker (Invitrogen).