Skip to main content
. 2007 Jan 5;3(2):91–99. doi: 10.7150/ijbs.3.91

Figure 3.

Figure 3

Screening for the U6-Neo-RNAi vector containing the insert and its expression in mouse after deletion of the neo by EIIa-Cre transgene. (A) After double-strand oligo ligation into the pBS/U6-neo vector, check insertion by SalI and EcoRI restriction and migration in a 3% agarose gel. Run two controls (parent and restricted parent vector) and an appropriate molecular weight marker (100, 150 and/or 200 bp). (B) Northern blot showing expression of pBSU6-Neo-RNAi against mouse Fgfr2. Cre mediated deletion of the neo from U6-ploxPneo-Fgfr2 transgene allows expression of Fgfr2 RNAi. (C) Assessment of copy number of U6-ploxPneo-Fgfr2 transgene by realtime PCR using primers for neo gene. Control DNA with 1 copy and 2 copies of neo gene are from Sirt6+/- and Fgfr1-/- ES cells, respectively. (D) RT-PCR analysis of Fgfr1-4 expression in E12.5 embryos of different genotypes. Wt: Wild type; U6: U6-ploxPneo-Fgfr2; Cre: EIIa-Cre; and U6;Cre: U6-Fgfr2;EIIa-Cre. The first row is a longer exposure of the second row. Three embryos for each genotype were shown. RT-PCR was performed using the following primers: Fgfr1-F: 5'-TTCTGGGCTGTGCTGGTCAC-3', and Fgfr1-R: 5'-GCGAACCTTGTAGCCTCCAA-3'. Fgfr2-F: 5'-AAGGTTTACAGCGATGCCCA-3', and Fgfr2-R: 5'-ACCACCATGCAGGCGATTAA-3'. Fgfr3-F: 5'-CTAGTGTTCTGCGTGGCGGT-3', and Fgfr3-R: 5'-TTCTTATCCATTCGCTCCGG-3'. Fgfr4-F: 5'-CTGTTGAGCATCTTTCAGGG-3', and Fgfr4-R: 5'-CGTGGAAGGCCTGTCCATCC-3'.