Transcript Gls2

Design and generation of a glutaminase Gls2 conditional
knockout mice
A.
1
Peñalver ,
1
Tosina ,
1
Martín-Rufián ,
1
Campos-Sandoval ,
1
Alonso ,
M.
M.
J. A.
F. J.
Segura1, J. M. Matés1, M. A. Ramírez2, A. Guitiérrez-Adán2, J. Márquez1
1Department
J. A.
of Molecular Biology and Biochemistry. Faculty of Science. University of Málaga.
Málaga (Spain)
2Department of Animal Reproduction, INIA. Madrid (Spain)
Materials and Methods
Introduction
 Generation of the Gls2 conditional knock-out mice
Mammalian glutaminase (GA; EC
3.5.1.2) 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 Gls2encoded 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.
Gls2tm2Mqz vector
Gls2tm2Mqz 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.
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)
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
Gls2tm2Mqz vector was linearized by enzymatic digestion with AsiSI and transfected by electroporation into B6D2F1 ES cells (electroporation conditions: 300V
during 500 μseg – room temperature).
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.
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.
Results
1. Embryonic stem cells (ESCs) culture
4. ESCs microinjection in 8-cell stage embryo
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.
B
A
Figure 5. (A) Embryo selection
based on their morphology. (B) ESCs
microinjection.
5. Chimeric pups
References
1.
2.
2. PCR screening of the transgenic ESCs
Martín-Rufián, M., et al. (2012). Mammalian
glutaminase Gls2 gene encodes two
functional alternative transcripts by a
surrogate promoter usage mechanism.
PlosOne: e38380.
Figure 3. PCR genotyping of
ESCs. ESCs transfected with
Gls2tm2Mqz transgenic vector
results in the amplication of a
207 bp band. (Gene Ruler 50
bp DNA Ladder shown in the
left)
ESCs transgenic samples
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: 1216-1222.
A
B
C
3.
8-cell stage embryos extraction
A
C
*This work was supported by Excellence Grant CVI6656 from the regional Government of Andalusia
and by Grant RD12/0028/0013 of the RTA RETICS
network from the Spanish Health Institute Carlos III.
Figure 6. (A) Chimeric pups obtained after transgenic ESCs
microinjection in Swiss embryos. Southern blot analysis (B) and threeprimer PCR strategy (C) were performed to guarantee the presence of
the vector.
Summary
B
Figure 4. (A) Oviducts extraction. (B) Microscopic image of the oviducts and
uterine horn. (C) Embryos located in the ampulla
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.