Skip to main content
. 2015 Oct 27;10(10):e0141611. doi: 10.1371/journal.pone.0141611

Fig 2. Generation of a stable klf2a sh317 mutant line.

Fig 2

(A) Schematic structure of wildtype klf2a gene with genomic sequence at the TALEN mutagenesis site. XcmI restriction site is indicated by the green line and the 19bp spacer for the TALEN structure of choice is indicated by the black line. The 14bp deletion identified in the klf2a sh317 allele is indicated by the sequence in red. (B) Schematic structure of Klf2a wildtype and Klf2a sh317 mutant proteins with amino acid (AA) lengths and predicted molecular weights in kilodaltons (kDa) Klf2a transactivation domain is in purple and transrepression domain is in yellow colour. (C) Substantial reduction of the 43kDa band representing the full-length Klf2a protein can be seen in a western blot of whole-embryo protein samples extracted from the F3 generation of klf2a sh317 mutant embryos at 5dpf, compared to wildtype embryos and heterozygous klf2a sh317 carriers. Additionally, two bands running at approximately 33kDa can be seen in the klf2a sh317 mutant, possibly representing truncated Klf2a. Asterixes indicate bands of approx. molecular weights of 38kDa and 47kDa consistent with expression of Klf4a and Biklf/Klf4b/Klf17 respectively, present in all three lanes in equal intensity. β actin was used as a loading control. (D) klf2a coding sequence was amplified from the control cDNA originating from 48hpf wildtype embryos giving an expected 1155bp PCR product. No band was detected in the sample with cDNA from unfertilised embryos (unf. cDNA) indicating the absence of maternal klf2a mRNA in unfertilised embryos. No 1674bp band was detected in unfertilised cDNA or control cDNA confirming absence of genomic DNA contamination. gapdh was used as a positive control. A band of expected size was detected in both the unfertilised eggs cDNA and control cDNA lanes indicating maternal gapdh mRNA in unfertilised zebrafish eggs. Abbreviations: -E: negative control with no reverse transcriptase added during reverse transcription (RT). -RNA: negative control with no RNA added during RT. B: blank, no template added to the PCR reaction. Abbreviations: L: Hyperladder II (NEB).