Figure 2.
TLK2 is an essential gene. (a) Schematic of the Tlk2 knockout-first allele (Tlk2T). The position of the genetrap cassette, the allelic configurations and their designations following FLP and CRE expression are shown. (b) Southern blot of SpeI-digested genomic DNA with a probe for neomycin to identify clones with single insertions. Six targeted clones and a negative control (C57BL/6) are shown and the expected size indicated. Additional Southern blots confirming correct targeting are shown in Supplementary Figure S1. (c) Tlk2 deficiency is embryonic lethal. The number of animals expected (exp.) from 27 Tlk2+/T breedings (112 pups total) assuming normal Mendelian inheritance is shown compared with the number of observed pups (obs.). Percentage observed is indicated above the bar graphs. Based on the genotypes of a total of 112 pups from 14 independent litters, a P-value of <0.0001 was determined using binomial distribution and indicated by ***. (d) Normal Mendelian distribution of genotypes was observed among 41 E10.5 to E13.5 embryos from six individual Tlk2+/T breedings. Percentage observed is indicated above the bar graphs. (e) Examples of littermate E10.5 embryos (scale bar=1 mm). (f) E12.5 embryos (scale bar=1 mm) (g) E15.5 embryos (scale bar=2 mm) and (h) E16.5 embryos (scale bar=2 mm). (i) Quantitative real-time PCR analysis of Tlk1 and Tlk2 levels in MEFs from littermates of the indicated genotype. The mean (red bars) and standard deviation (S.D.) of three replicates are plotted. (j) Western blotting of TLK1 and TLK2 protein levels in MEFs of the indicated genotype. Histone H3 is shown as a loading control and a nonspecific (NS) band recognized by the TLK2 antibody is indicated. (k) Hematoxylin and eosin (H&E) (left panels) or Ki67 immunohistochemical staining (right panels) of E12.5 embryo sections of the indicated genotype (scale bar=1 mm)