4.ESCs microinjection in 8-cell stage embryo 5. Chimeric pups Design and generation of a glutaminase Gls2 conditional knockout mice A. Peñalver 1, M. Tosina 1, M. Martín-Rufián 1, J. A. Campos-Sandoval 1, F. J. Alonso 1, J. A. Segura 1, J. M. Matés 1, M. A. Ramírez 2, A. Guitiérrez-Adán 2, J. Márquez 1 1 Department of Molecular Biology and Biochemistry. Faculty of Science. University of Málaga. Málaga (Spain) 2 Department of Animal Reproduction, INIA. Madrid (Spain) Materials and Methods References Introduction Mammalian glutaminase (GA; EC ) is the main enzyme involved in brain generation of glutamate (Glu). This amino acid acts as an excitatory neurotransmitter within the CNS, and it is also implicated in behavioral sensitization through the mesolimbic pathway. Two different GA genes have been described: Gls that encodes the isozymes KGA and GAC, and Gls2, which encodes GAB and LGA isozymes. Gls and Gls2 isoforms are co-expressed in different brain regions and cells. Of note, location of Gls2- encoded isoforms in neuronal nuclei suggests a novel role in transcriptional regulation. The functional role of different GA isoforms in mammalian brain is so far unexplained. We aim to elucidate the cerebral function of Gls2; for this purpose, we obtained a transgenic vector carrying the Gls2 gene between loxP sites (acquired from the EUCOMM consortium), which leads to a conditional knock-out (KO) mouse model. Based on the Cre–recombinase system this model will allow silencing of Gls2 isoforms in the main glutamatergic regions of the brain.  Generation of the Gls2 conditional knock-out mice Gls2 tm2Mqz vector *This work was supported by Excellence Grant CVI from the regional Government of Andalusia and by Grant RD12/0028/0013 of the RTA RETICS network from the Spanish Health Institute Carlos III. Gls2 tm2Mqz vector was obtained from the EUCOMM consortium. It was designed using the R3R4_pBR_DTA+_Bsd_amp backbone, where the Gls2 sequence (exon 1 to 12) was inserted. Two homology arms flank a positive drug selection marker (neo). A negative selection marker (DTA) is placed adjacent to one of the targeting arms. A unique restriction enzyme site is located between the vector backbone and the homology arm (AsiSI) to linearize the vector. The long homology arm (5’ arm-5377 bp) corresponds to exon and intron 1 of the Gls2 genomic sequence. The short arm (3’ arm-3914 bp) shares homology with exons 8 to 12. Both arms flank the region to be deleted from the Gls2 target gene (exons 2 to 7), result of the Cre-recombinase action, which will excise the region between loxP sites. The incorporation of this transgenic vector into the murine embryonic stem cells (ES) will entail the generation of Gls2 conditional KO mouse. Generation of Gls2 mutant mice Gls2 tm2Mqz vector was linearized by enzymatic digestion with AsiSI and transfected by electroporation into B6D2F1 ES cells (electroporation conditions: 300V during 500 μseg – room temperature). Transgenic ES cells were selected by geneticin (150 μg/ml) and PCR-genotyped before their microinjection in 8-cell stage embryos (Swiss strain). These embryos were obtained by superovulation of mice. Swiss female mice were superovulated with equine chorionic gonadotropin (PMSG) and human chorionic gonadotropin (HCG), inmediately after HCG injection, female mice were mated 1:1 to male mice (same strain). We checked female mice for copulation plugs to proceed to embryo extraction. Embryo implantation was performed in pseudopregnant state mice (pseudopregnancy was induced by mating with vasectomized males). After 20 days chimeric pups were born. The chimeric pups carrying the modification within their germ line were use to generate the homozygous Gls2 (-/-) mice. After integration of the vector in both alleles, the mice will be mated with mutant Cre mice, which express this recombinase enzyme under control of the synapsin promoter. This will result in a deletion of the exons 2 to 7 giving rise to null Gls2 mutants mainly in the following brain areas: cortex, hippocampus, amygdala and cerebellum, which are essential for glutamatergic transmission and related to the mesolimbic pathway. Figure 1. Gls2 tm2Mqz vector. Schematic representation of the targeting vector. The main features of the vector are indicated below. (Image obtained from EUCOMM website) Results 1.Embryonic stem cells (ESCs) culture 2.PCR screening of the transgenic ESCs 3. 8-cell stage embryos extraction Figure 2. Microscopic image from ESCs culture (B6D2F1 strain). Feeder cells (129/Sv strain) are essential to the culture and sustaining of undifferentiated ESCs. Feeder cells were prepared using mitomycin-C. ESCs transgenic samples Vector DNA H20H20 Figure 3. PCR genotyping of ESCs. ESCs transfected with Gls2 tm2Mqz transgenic vector results in the amplication of a 207 bp band. (Gene Ruler 50 bp DNA Ladder shown in the left) Figure 4. (A) Oviducts extraction. (B) Microscopic image of the oviducts and uterine horn. (C) Embryos located in the ampulla A B C 1. Martín-Rufián, M., et al. (2012). Mammalian glutaminase Gls2 gene encodes two functional alternative transcripts by a surrogate promoter usage mechanism. PlosOne: e Ramírez, M.A., et al. Effect of stem cell activation, culture media of manipulated embryos, and site of embryo transfer in the production of Fo embryonic stem cell mice. Biol. Reprod. 80: Figure 5. (A) Embryo selection based on their morphology. (B) ESCs microinjection. A B Figure 6. (A) Chimeric pups obtained after transgenic ESCs microinjection in Swiss embryos. Southern blot analysis (B) and three- primer PCR strategy (C) were performed to guarantee the presence of the vector. Summary Obtaining homozygous mice will allow us to create a Gls2 conditional knock-out mouse (knocking down both LGA and GAB isoforms) after mating these animals with mice expressing Cre-recombinase under control of a specific tissue promoter. A DNA Vector H2OH2O C B