Establishment and Identification of the Neocortex and Hippocampus <i>GABRG2</i> Knockout Mice and Its Preliminary Study in Generalized Epilepsy with Febrile Seizures Plus

doi:10.13523/j.cb.1908034

China Biotechnology

2020, Vol. 40

Issue (3): 9-20 DOI: 10.13523/j.cb.1908034

Orginal Article

Establishment and Identification of the Neocortex and Hippocampus GABRG2 Knockout Mice and Its Preliminary Study in Generalized Epilepsy with Febrile Seizures Plus

GUO Sheng-nan^1,^2,^**,LI Xin-xiao^2,^3,^**,WANG Feng^2,³,LIU Kun-mei²,DING Na²,HU Qi-kuan^1,^2,^***(

),SUN Tao^2,^3,^***(

)

1 Department of Physiology, Basic Medical School of Ningxia Medical University, Yinchuan 750004, China
2 Ningxia Key Laboratory of Cerebrocranial Diseases, Yinchuan 750004, China
3 Department of Neurosurgery, General hospital of Ningxia Medical University, Yinchuan 750004, China

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Abstract

To establish and identify the neocortex and hippocampus specific GABA_A receptor γ2 subunit (GABRG2) knockout mice via Cre/Loxp conditional gene knockout technology, and provide the important animal model for further investigating the functional role of GABRG2 in hippocampus and neocortex GABRG2 in epileptogenesis. The constructed GABRG2 ^fl/wt mice and the mice that express Cre recombinase to approximately 88% of the neurons of the neocortex and hippocampus were bred and authenticated, respectively. Mating and identification of GABRG2 ^fl/fl mice with Cre mice were carried out, and the GABRG2 ^fl/wt Cre ⁺ mice were screened. The mouse genotypes were identified by PCR. The Real-Time PCR and Western blot were used to detect the expression of GABRG2 mRNA and protein levels in mouse neocortex and hippocampus. PCR results indicate that mouse genotypes are consistent with GABRG2 ^fl/wt Cre ⁺. Compared with control group mice, GABRG2 mRNA and protein levels were significantly reduced. During the temperature elevation, the GABRG2 ^fl/wt Cre ⁺ mice had significant seizures. Based on the Cre/loxp conditional gene knockout technology, that succeed in building a group of neocortex and hippocampus specific GABRG2 gene knockout mice which can stably go down to the future generation, which would supply a technical basis for animal models in further researches of the regulation and mechanism of GABRG2 gene in the progress of epilepsy.

Key words： GABRG2 Genetic epilepsy with ebrile eizures plus Neocortex and hippocampus Cre/Loxp recombination system Gene knockout

Received: 20 August 2019 Published: 18 April 2020

ZTFLH:

Q78

Corresponding Authors: Qi-kuan HU,Tao SUN E-mail: huqikuan@163.com;suntao6699@163.com

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	GUO Sheng-nan
	LI Xin-xiao
	WANG Feng
	LIU Kun-mei
	DING Na
	HU Qi-kuan
	SUN Tao

Cite this article:

GUO Sheng-nan, LI Xin-xiao, WANG Feng, LIU Kun-mei, DING Na, HU Qi-kuan, SUN Tao. Establishment and Identification of the Neocortex and Hippocampus GABRG2 Knockout Mice and Its Preliminary Study in Generalized Epilepsy with Febrile Seizures Plus. China Biotechnology, 2020, 40(3): 9-20.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1908034 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I3/9

Fig.1 Strategy of hippocampus and neocortex GABRG2 knockout mice (a)Donor vector and CRISPR/Cas9 system (b)Atlas of targeted vector

Fig.2 Schematic design of primers for GABRG2 flox mouse identification

Table1 Primer information for GABRG2 flox mouse identification

Table 2 Primer information for GABRG2^fl/wtCre⁺ mouse identification

Fig.3 PCR genotyping results of the offspring of F₀ mice cross with C57BL/6J mice (a) PCR genotyping results of the primer 1 revealed two bands, which one is 248bp, the other is 158bp (b) PCR genotyping results of the primer 2 also revealed two bands, which one is 294bp and the other is 201bp (c) Results of primer 3 PCR indicted that there is a band containing the flox site, and no band without the flox site (d) Results of primer 4 PCR indicted that there is a band containing the flox site, and no band without the flox site (e) 5'allele was detected by PCR amplification, the agarose gel electrophoresis results that there is a 1 962bp band containing the flox site, and no band without the flox site (f) 3'allele was also detected by PCR amplification, the agarose gel electrophoresis results that there is a 2 013bp band containing the flox site, and no band without the flox site M: Marker;1-4:GABRG2^fl/wt; 5:WT;6:H₂O

Fig.4 PCR genotyping results of the Cre mice (a) PCR genotyping results of the wild type primer revealed that the wild type mice have a band, the mutant mice are not (b) Results of mutant primer PCR indicted that there is a band in mutant mice, the wild type mice are not M: Marker;1-6:Cre^+/+ mice;7:WT;8:H₂O

Fig.5 PCR genotyping results of the offspring of GABRG2^fl/wtCre^+/- mice and GABRG2^fl/wtCre^+/- mice (a) PCR genotyping results of the primer 1 revealed two bands, which one is 248bp, the other is 158bp (b) Results of primer 2 PCR indicted that there is a band containing the flox site, and no band without the flox site (c) Primer 3 was used to detect the Cre-positive mice by PCR, results suggested that there is a 481bp band (4) Primer 4 was used to detect the null site by PCR, results suggested that there is a 424bp band, 1#, 14#, 15# represent heterozygotes, homozygotes, and wild type, respectively M:Marker;H:Hippocampus;C:Neocortex;1-6,8-13:GABRG2^fl/wtCre⁺ mice;7,15:Wild type mice;14:GABRG2^fl/flCre⁺ mouse;16:H₂O

Fig.6 Evaluation of GABRG2 protein expression in NC-GABRG2 group and KO-GABRG2 group (a) Western blot verified the protein expression level of GABRG2 (b) GraphPad prism 5.0 software analyzed the results of Western blot *** P< 0.001 vs normal control (NC) group H: Hippocampus; C: Neocortex; NC: Normal control; KO: Knock out

Fig.7 The electroencephalography electrode implant surgery (a) The electrode and circular sleeve (b) Maintenance of anesthesia with 2% isoflurane (c) An incision was made on the scalp to expose the skull (d) Three holes were drilled through the skull to dura, one placed 1-2mm left laterosuperior to the bregma and two placed 2-3mm poster to the bregma, each being 2-3mm lateral to the central sulcus (e) Two epidural electrodes and one stainless steel screw were carefully implanted (f) Electrodes and screw were fixed by dental cement ◇: Bregma region

Fig.8 GABRG2^fl/wtCre⁺ mice but not normal control group have seizures induced by elevated body core temperature (a) The temperature induction apparatus and heating setup (b) The number of jumping bouts during temperature induction was plotted for NC group and KO mice (c) The initial temperature of the first jumping was plotted for NC group and KO mice (d) Comparison of the rate of core body temperature change assessed over 2min intervals during temperature induction between NC mice and KO mice (e) Representative 5s long traces of intracranial EEG activity at the temperature specified at the top of the color code, and no seizures were seen up to 42.5℃ in any NC mice (f) The intracranial EEG activity at a specified temperature in KO mice, and a seizure was provoked at 39℃ or 39.5℃ (n=16 for NC group; n=16 for KO group)


[1]	Kumamoto E . The pharmacology of amino-acid responses in septal neurons. Prog Neurobiol, 1997,52(3):197-259.

[2]	Whiting P J . The GABA-A receptor gene family: new targets for therapeutic intervention. Neurochem Int, 1999,34(5):387-390.

[3]	Laurie D J, Wisden W, Seeburg P H . The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. J Neurosci, 1992,12(11):4151-4172.

[4]	Asarnow R F ,LoPresti C, Guthrie D, et al. Developmental outcomes in children receiving resection surgery for medically intractable infantile spasms. Dev Med Child Neurol, 1997,39(7):430-440.

[5]	Daniel R T, Meagher-Villemure K, Roulet E , et al. Surgical treatment of temporoparietooccipital cortical dysplasia in infants: report of two cases. Epilepsia, 2004,45(7):872-876.

[6]	Duchowny M, Jayakar P, Resnick T , et al. Epilepsy surgery in the first three years of life. Epilepsia, 1998,39(7):737-743.

[7]	Duchowny M S, Resnick T J, Alvarez L A , et al. Focal resection for malignant partial seizures in infancy. Neurology, 1990,40(6):980-984.

[8]	Duncan J S, Sander J W, Sisodiya S M , et al. Adult epilepsy. Lancet, 2006,367(9516):1087-1100.

[9]	Kwan P, Brodie M J . Neuropsychological effects of epilepsy and antiepileptic drugs. Lancet, 2001,357(9251):216-22.

[10]	Bell G S, Neligan A, Sander J W . An unknown quantity-the worldwide prevalence of epilepsy. Epilepsia, 2014,55(7):958-962.

[11]	Espinosa-Jovel C, Toledano R, Aledo-Serrano á , et al. Epidemiological profile of epilepsy in low income populations. Seizure, 2018,56:67-72.

[12]	Dua T, de Boer H M, Prilipko LL , et al. Epilepsy care in the world: results of an ILAE/IBE/WHO global campaign against epilepsy survey. Epilepsia, 2006,47(7):1225-1231.

[13]	Sadr S S, Javanbakht J, Javidan A N , et al. Descriptive epidemiology: prevalence, incidence, sociodemographic factors, socioeconomic domains, and quality of life of epilepsy: an update and systematic review. Arch Med Sci, 2018,14(4):717-724.

[14]	Trinka E, Kwan P, Lee B , et al. Epilepsy in Asia: Disease burden, management barriers, and challenges. Epilepsia, 2019,60(Suppl 1):7-21.

[15]	Song P, Liu Y, Yu X , et al. Prevalence of epilepsy in China between 1990 and 2015: A systematic review and meta-analysis. J Glob Health, 2017,7(2):020706.

[16]	Yu Z, Dong K, Chang H , et al. The epidemiological and clinical characteristics study on epilepsy in 8 ethnic groups of China. Epilepsy Res, 2017,138:110-115.

[17]	Baulac S, Huberfeld G, Gourfinkel-An I , et al. First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene. Nat Genet, 2001,28(1):46-48.

[18]	Harkin L A, Bowser D N, Dibbens L M , et al. Truncation of the GABAA-receptor γ2 subunit in a family with generalized epilepsy with febrile seizures plus. Am J Hum Genet, 2002,70(2):530-536.

[19]	Audenaert D, Schwartz E, Claeys K G , et al. A novel GABRG2 mutation associated with febrile seizures. Neurology, 2006,67(4):687-690.

[20]	Frugier G, Coussen F, Giraud M F , et al. A gamma 2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface. J Biol Chem, 2007,282(6):3819-3828.

[21]	Lachance-Touchette P, Brown P, Meloche C , et al. Novel α1 and γ2 GABAA receptor subunit mutations in families with idiopathic generalized epilepsy. Eur J Neurosci, 2011,34(2):237-249.

[22]	Kananura C, Haug K, Sander T , et al. A splice-site mutation in GABRG2 associated with childhood absence epilepsy and febrile convulsions. Arch Neurol, 2002,59(7):1137-1141.

[23]	Sun H, Zhang Y, Liang J , et al. SCN1A, SCN1B, and GABRG2 gene mutation analysis in Chinese families with generalized epilepsy with febrile seizures plus. J Hum Genet, 2008,53(8):769-674.

[24]	Kang J Q, Shen W, Lee M , et al. Slow degradation and aggregation in vitro of mutant GABAA receptor gamma2(Q351X) subunits associated with epilepsy. J Neurosci, 2010,30(41):13895-13905.

[25]	Hernandez C C, Gurba K N, Hu N , et al. The GABRA6 mutation, R46W, associated with childhood absence epilepsy, alters α6β2γ2 and α6β2δ GABAA receptor channel gating and expression. J Physiol, 2011,589(Pt 23):5857-5878.

[26]	Ishii A, Kanaumi T, Sohda M , et al. Association of nonsense mutation in GABRG2 with abnormal trafficking of GABAA receptors in severe epilepsy. Epilepsy Res, 2014,108(3):420-432.

[27]	Orenstein N, Goldberg-Stern H, Straussberg R , et al. A de novo GABRA2 missense mutation in severe early-onset epileptic encephalopathy with a choreiform movement disorder. Eur J Paediatr Neurol, 2018,22(3):516-524.

[28]	Shen D, Hernandez C C, Shen W , et al. De novo GABRG2 mutations associated with epileptic encephalopathies. Brain, 2017,140(1):49-67.

[29]	Tian M, Mei D, Freri E , et al. Impaired surface αβγ GABAA receptor expression in familial epilepsy due to a GABRG2 frameshift mutation. Neurobiol Dis, 2013,50:135-141.

[30]	Sancar F, Czajkowski C . A GABAA receptor mutation linked to human epilepsy (γ2R43Q) impairs cell surface expression of αβγ receptors. J Biol Chem, 2004,279(45):47034-47039.

[31]	Kang J Q, Shen W, Zhou C , et al. The human epilepsy mutation GABRG2(Q390X) causes chronic subunit accumulation and neurodegeneration. Nat Neurosci, 2015,18(7):988-996.

[32]	Balan S, Sathyan S, Radha S K , et al. GABRG2, rs211037 is associated with epilepsy susceptibility, but not with antiepileptic drug resistance and febrile seizures. Pharmacogenet Genomics, 2013,23(11):605-610.

[33]	Butil? A T, Zazgyva A, Sin A I , et al. GABRG2 C588T gene polymorphisms might be a predictive genetic marker of febrile seizures and generalized recurrent seizures: a case-control study in a Romanian pediatric population. Arch Med Sci, 2018,14(1):157-166.

[34]	Abou El Ella S S, Tawfik M A ,Abo El Fotoh W M M, et al. The genetic variant "C588T" of GABARG2 is linked to childhood idiopathic generalized epilepsy and resistance to antiepileptic drugs. Seizure, 2018,60:39-43.

[35]	Kang J Q, Shen W, Macdonald R L . Why does fever trigger febrile seizures? GABAA receptor gamma2 subunit mutations associated with idiopathic generalized epilepsies have temperature-dependent trafficking deficiencies. J Neurosci, 2006,26(9):2590-2597.

[36]	Chandra D, Korpi E R, Miralles C P , et al. GABAA receptor gamma 2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines. BMC Neurosci, 2005,6:30.

[37]	Oakley J C, Kalume F, Yu F H , et al. Temperature-and age-dependent seizures in a mouse model of severe myoclonic epilepsy in infancy. Proc Natl Acad Sci USA, 2009,106(10):3994-3999.

[38]	Han S, Tai C, Westenbroek R E , et al. Autistic-like behaviour in Scn1a +/- mice and rescue by enhanced GABA-mediated neurotransmission . Nature, 2012,489(7416):385-390.

[39]	King S M . Escape-related behaviours in an unstable elevated and exposed environment. I. A new behavioural model of extreme anxiety. Behav Brain Res, 1999,98(1):113-126.

[40]	Warner T A, Liu Z, Macdonald R L , et al. Heat induced temperature dysregulation and seizures in Dravet Syndrome/GEFS+ Gabrg2+/Q390X mice. Epilepsy Res, 2017,134:1-8.

[41]	Wallace R H, Marini C, Petrou S , et al. Mutant GABAA receptor γ2-subunit in childhood absense epilepsy and febrile seizures. Nature genetics, 2001,28(1) : 49-52.

[42]	Wang X M, Xu M C, Du L Z . Association analysis of gamma2 subunit of gamma-aminobutyric acid (GABA) type A receptor and voltage-gated sodium channel type II alpha-polypeptide gene mutation in southern Chinese children with febrile seizures. J Child Neurol, 2007,22(6):714-719.

[43]	Hindocha N, Nabbout R, Elmslie F , et al. A case report of a family with overlapping features of autosomal dominant febrile seizures and GEFS+. Epilepsia, 2009,50(4):937-942.

[44]	Salam S M, Rahman H M, Karam R A . GABRG2 gene polymorphisms in Egyptian children with simple febrile seizures. Indian J Pediatr, 2012,79(11):1514-1516.

[45]	Hung K L, Liang J S, Wang J S , et al. Association of a novel GABRG2 splicing variation and a PTGS2/COX-2 single nucleotide polymorphism with Taiwanese febrile seizures. Epilepsy Res, 2017,129:1-7.

[46]	Ito M, Yamakawa K, Sugawara T , et al. Phenotypes and genotypes in epilepsy with febrile seizures plus. Epilepsy Res, 2006,70(Suppl1):S199-205.

[47]	Kos C H . Cre/loxP system for generating tissue-specific knockout mouse models. Nutr Rev, 2004,62(6Pt1):243-246.

[48]	Schmidt-Supprian M, Rajewsky K . Vagaries of conditional gene targeting. Nat Immunol, 2007,8(7):665-668.

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