Transcript Gls2
Introduction
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 which encodes GAB and isozymes. Gls and Gls2 isoforms are co-expressed in different brain regions and cells. Of note, location of
Gls2
, LGA
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 consortium), from which the EUCOMM leads to a conditional knock-out model. Based on the (KO) mouse
Cre
–recombinase system this model will allow silencing of
Gls2
isoforms in the glutamatergic regions of the brain.
main
Design and generation of a glutaminase
Gls2
knockout mice conditional
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
Generation of the Gls2 conditional knock-out mice
Gls2 tm2Mqz
vector
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)
Gls2
tm2Mqz
was vector was obtained from the EUCOMM consortium. It 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 excise the region between
Cre loxP
-recombinase action, which will 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 during 500 μseg – room temperature).
B6D2F1
ES cells (electroporation conditions: 300V Transgenic ES cells were selected by geneticin (150 embryos were obtained by superovulation of mice.
μg/ml) and PCR-genotyped before their microinjection in 8-cell stage embryos (
Swiss Swiss
strain). These 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 alleles, the mice will be mated with mutant
Cre Gls2
(-/-) mice. After integration of the vector in both 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.
A B
Figure 5.
(A) Embryo selection based on their morphology. (B) ESCs microinjection.
5. Chimeric pups
References 1.
Martín-Rufián, M.,
glutaminase Gls2 et al. (2012)
.
gene Mammalian encodes two functional surrogate alternative promoter
PlosOne: e38380.
transcripts usage by a mechanism.
2.
Ramírez, M.A.,
activation, et al. Effect of stem cell culture media of manipulated embryos, and site of embryo transfer in the production of Fo embryonic stem cell mice.
Biol. Reprod. 80: 1216-1222.
2. PCR screening of the transgenic ESCs ESCs transgenic samples
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
left) shown in the
C A B
*This work was supported by Excellence Grant CVI 6656 from the regional Government of Andalusia and by Grant RD12/0028/0013 of the RTA RETICS network from the Spanish Health Institute Carlos III.
3.
8-cell stage embryos extraction
A B
Figure 4. (A) Oviducts extraction. (B) Microscopic image of the oviducts and uterine horn. (C) Embryos located in the ampulla
C
Figure 6.
(A) microinjection in Chimeric
Swiss
pups obtained after transgenic ESCs 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