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. 2016 Jan 8;9:165. doi: 10.3389/fnsys.2015.00165

Figure 1.

Figure 1

Generation of conditional floxGLS1 mice. (A) Insertion of 3-loxP construct into the GLS1 gene. (A1) A targeting construct engineered with loxP sites flanking exon 1 (thick black line with transcription start ATG codon indicated) of the GLS1 gene encoding glutaminase was inserted into the endogenous GLS1 locus by homologous recombination. The targeting vector (green) also contained a floxed PGK-neo-cassette (thick blue line) for positive selection of the ES cell clones. The blue bar indicates the position of external Probe A (blue), used for Southern analysis, while the black arrow indicates the length of the expected wild-type band and the red arrow the length of the expected mutant band. Note that an additional HindIII site was introduced along with the middle loxP site. (A2) In a HindIII digest of ES cell DNA, Probe A hybridized to a 6.5 kb band for the WT allele, in contrast to a 5.0 kb band for the 3-loxP allele. Results are shown for ES cells with non-homologous recombination (NHR) and homologous recombination (HR). (B) Breeding floxGLS1 mouse. (B1) 3-loxP mice were crossed with EIIacre partial deletor mice to remove the floxed PGK-neo cassette. 2-loxP progeny were identified by PCR genotyping using two primer pairs flanking the loxP sites. Blue bars indicate the location of the primers. (B2) The PCR gels show genotyping results for two mice. In the top gel, the 5′ loxP site was revealed by a 214 bp band, in addition to the WT allele of 157 bp in the mutant mouse (2-loxP). In the bottom gel, the 3′ loxP site was revealed by a 430 bp band, in addition to the WT and of 341 bp in the mutant mouse. (C) GLS1 mRNA expression from the floxGLS1 allele. mRNA was measured in whole hippocampus (Hipp) and frontal cortex (FC) tissue. Relative expression was normalized to the corresponding WT mice (dashed line), done in two cohorts of mice, lox/+ and lox/lox mice. There was no genotypic impact of GLS1 flox status on GLS1 mRNA. The number of samples is indicated above the bars (gray numbers correspond to the lox/+ cohort and black numbers correspond the lox/lox cohort).