HHS Public Access Author manuscript Author Manuscript
Epilepsia. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Epilepsia. 2016 June ; 57(6): e103–e107. doi:10.1111/epi.13390.
Cacna1g is a genetic modifier of epilepsy caused by mutation of voltage-gated sodium channel Scn2a Jeffrey D. Calhoun1,2,*, Nicole A. Hawkins1,3,*, Nicole J. Zachwieja1, and Jennifer A. Kearney1,2,† 1Department
of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, 60611, USA
Author Manuscript
2Department
of Medicine, Vanderbilt University, Nashville, Tennessee, 37232, USA
3Neuroscience
Program, Vanderbilt University, Nashville, Tennessee, 37232, USA
Summary
Author Manuscript
More than 1200 mutations in neuronal voltage-gated sodium channel genes have been identified in patients with several epilepsy syndromes. A common feature of genetic epilepsies is variable expressivity among individuals with the same mutation. The Scn2aQ54 transgenic mouse model has a mutation in Scn2a that results in spontaneous epilepsy. Scn2aQ54 phenotype severity varies depending on the genetic strain background, making it a useful model for identifying and characterizing epilepsy modifier genes. Scn2aQ54 mice on the [C57BL/6JxSJL/J]F1 background exhibit earlier seizure onset, elevated spontaneous seizure frequency, and decreased survival compared to Scn2aQ54 mice congenic on the C57BL/6J strain. Genetic mapping and RNA-Seq analysis identified Cacna1g as a candidate modifier gene at the Moe1 locus, which influences Scn2aQ54 phenotype severity. In this study, we evaluated the modifier potential of Cacna1g, encoding the Cav3.1 voltage-gated calcium channel, by testing whether transgenic alteration of Cacna1g expression modifies severity of the Scn2aQ54 seizure phenotype. Scn2aQ54 mice exhibited increased spontaneous seizure frequency with elevated Cacna1g expression and decreased seizure frequency with decreased Cacna1g expression. These results provide support for Cacna1g as an epilepsy modifier gene and suggest that modulation of Cav3.1 may be an effective therapeutic strategy.
Keywords
Author Manuscript
Seizures; Mouse model; Voltage-gated ion channels; Voltage-gated calcium channels; Genetics
†
Corresponding Author Jennifer A. Kearney, Department of Pharmacology, Searle 8-510, 320 East Superior St. Northwestern University, Chicago, IL 60611, Tel: 312-503-4894, [email protected]. *indicates equal contribution Disclosure None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
Calhoun et al.
Page 2
Author Manuscript
Introduction Mutations in the voltage-gated sodium channel (VGSC) genes SCN1A, SCN2A, SCN3A, and SCN8A are responsible for several human epilepsies. More than 1200 mutations in SCN1A, SCN2A, and SCN8A have been identified in patients with various epilepsy syndromes, including Genetic Epilepsy with Febrile Seizures Plus, Benign Familial Neonatal-Infantile Seizures, and early infantile epileptic encephalopathies. Variable expressivity is a common feature of epilepsies caused by VGSC mutations.
Author Manuscript
Mouse epilepsy models provide a tractable system to map and characterize modifier genes. A common feature of these models is strain-dependent phenotype severity, suggesting the contribution of genetic modifiers. Scn2aQ54 (Q54) transgenic mice have a gain-of-function mutation that results in spontaneous focal motor seizures and strain-dependent epilepsy severity.1; 2 On the C57BL/6J (B6) strain, the Q54 mutation (B6.Q54) results in mild, adultonset epilepsy. When B6.Q54 is crossed to SJL/J (SJL), the resulting F1.Q54 mice experience juvenile-onset spontaneous seizures and reduced lifespan. We previously mapped modifier loci responsible for the strain difference in seizure frequency exhibited by Scn2aQ54 mice: Moe1 (modifier of epilepsy 1) and Moe2 on chromosome 11 and 19, respectively.2 Kcnv2 was identified as a modifier gene at Moe2.3 Recent work indicated multiple genes likely underlie the Moe1 peak, including Cacna1g and Hlf.4 Genetic abalation of Hlf, encoding a PAR bZIP transcription factor, exacerbated epilepsy in the Scn2aQ54 and Scn1a+/− Dravet mouse models.5
Author Manuscript
Cacna1g resides within the Moe1 locus and RNA-seq showed strain-dependent differences in Cacna1g transcript expression and alternative splicing.4 Cacna1g encodes the poreforming α1G/Cav3.1 subunit, a low-voltage activated calcium channel of the T-type calcium channel family. Patient and animal model data have demonstrated a link between T-type currents and absence seizures. Ethosuximide (ETX), a T-type channel blocker, is widely used for treatment of absence epilepsy in patients. In mice, targeted deletion of Cacna1g diminishes GABAB-receptor agonist-induced spike-wave discharges (SWDs), while overexpression of Cacna1g is associated with absence seizures and abnormal thalamocortical synchronization.6–8 A role for Cacna1g in non-absence epilepsy has been remained elusive. This study evaluated the modifier effect of Cacna1g on the Scn2aQ54 mouse epilepsy model. We tested the effect of transgenic upregulation or heterozygous deletion of Cacna1g on spontaneous seizure frequency of Scn2aQ54 mice.
Author Manuscript
Methods Mice A B6-derived BAC clone containing Cacna1g (RP23-65I14) was obtained from BACPAC Resources (http://bacpac.chori.org/home.htm). Pronuclear injections into fertilized SJL or B6 oocytes were performed by the Vanderbilt University Transgenic Mouse/ESC Shared Resource and the University of Michigan Transgenic Animal Model Core, respectively. Injections resulted in two Cacna1g BAC transgenic founders: (1) SJL.Cacna1gLOW (SJL.
Epilepsia. Author manuscript; available in PMC 2017 June 01.
Calhoun et al.
Page 3
Author Manuscript
1GL), maintained by continued backcrossing of hemizygous transgenic mice to SJL/J (Jackson Labs); and (2) B6.Cacna1gHIGH (B6.1GH), maintained by continued backcrossing of hemizygous transgenic mice to C57BL/6J (Jackson Labs). B6(129S4)Cacna1gtm1Stl/J mice were obtained (Jackson Labs #021932), bred to C57BL/6-Tg(Zp3cre)93Knw/J (Jackson Labs #003651), and maintained by continued backcrossing of heterozygous Cacna1gKO/+ (B6.1GKO/+) offspring to B6.Scn2aQ54 transgenic mice [Tg(Eno2-Scn2a1*)Q54Mm] congenic on B6 (B6.Q54) were maintained by continued backcrossing of hemizygous B6.Q54 males to B6 females.1
Author Manuscript
Transgenic SJL.1GL (SJL/J background) and B6.1GH (C57BL/6J background) females were crossed with B6.Q54 males to generate double transgenic (F1.Q54;1GL or B6.Q54;1GH) mice and single transgenic littermate controls. B6.1GKO/+ females were crossed to B6.Q54 mice, and B6.Q54;1GKO/+ male offspring were subsequently crossed to SJL/J females to generate F1.Q54;1GKO/+ mice and single transgenic littermate controls. Mice were group-housed with access to food and water ad libitum. All studies were approved by the Vanderbilt University and Northwestern University Animal Care and Use Committees in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The principles outlined in the ARRIVE guideline and Basel declaration (including the 3R concept) were considered when planning the experiments. Genotyping
Author Manuscript
Mice were genotyped by PCR of DNA isolated from postnatal day 14 (P14) tail biopsies. SJL.1GL and B6.1GH transgenic mice were identified using 1G-SP6 and 1G-T7 primers that amplify 200 bp and 220 bp products, respectively. The Q54 transgene and 1GKO/+ allele were genotyped as described.1; 9 Primer sequences used for genotyping, BAC integrity, copy number, and expression analysis will be provided upon request. BAC Integrity and Copy Number BAC integrity was evaluated by PCR amplification of genomic DNA. SP6 and T7 promoters flanked each end of the BAC insert, while microsatellite markers were located approximately every 32 Kb, including two within Cacna1g. B6.1GH mice were backcrossed twice to SJL to obtain informative mice for integrity analysis of the B6-derived BAC.
Author Manuscript
Transgene copy number was assessed by quantitative PCR (qPCR) using Cacna1g and reference Scn5a Taqman assays previously described.8; 10 At least n>4 independent DNA samples from hemizygous transgenics and wildtype controls were used to estimate copy number. Standard curves were generated by spiking SJL or B6 genomic DNA with BAC plasmid DNA equivalent to 0, 5, 10 and 20 copies per diploid genome. Transcript Analysis and Protein Analysis RNA was extracted from whole brains of six week old mice using Trizol (Life Technologies). The six week time point was selected to match our previous RNA-seq transcriptome analysis that identified Cacna1g as a candidate modifier.4 Total RNA was reverse transcribed using SuperScript III (Life Technologies). Quantitative digital droplet
Epilepsia. Author manuscript; available in PMC 2017 June 01.
Calhoun et al.
Page 4
Author Manuscript
PCR (ddPCR) was performed using ddPCR Supermix for Probes (No dUTP) (Bio-Rad) and TaqMan Gene Expression Assays (Life Technologies) for Cacna1g (FAMMm00486571_m1) and TATA binding protein (Tbp) (VIC-Mm00446971_m1). Reactions were partitioned into 20,000 droplets (1 nl each) in a QX200 droplet generator (Bio-Rad). Thermocycling conditions were 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute (ramp rate of 2°C/sec), and a final inactivation step of 98°C for 10 minutes. Following amplification, droplets were analyzed with a QX200 droplet reader and QuantaSoft v1.6.6.0320 software (Bio-Rad). All assays lacked detectable signal in no-RT and no-template controls. Relative transcript levels were expressed as a ratio of Cacna1g/Tbp concentration and compared between groups by Student’s T-test.
Author Manuscript
Membrane protein fractions (50 μg) were prepared from whole brains of six week old mice and separated on 4–20% gradient SDS-PAGE gels (Biorad) and transferred to nitrocellulose. Membranes were assayed using mouse monoclonal Cav3.1 (Neuromab, N178A/9; 1:500) and β-tubulin (Sigma, TUB 2.1; 1:2,000) primary antibodies, and peroxidase-conjugated goat anti-mouse (1:50,000; Jackson ImmunoResearch) secondary antibodies. Signals were detected with SuperSignal West Femto (Pierce) and densitometry was performed using ImageJ. Cav3.1 expression was normalized to the corresponding β-tubulin band. Expression differences were compared between groups by Student’s T-test. Phenotyping
Author Manuscript
Transgenic mice were phenotyped as previously described.4 Briefly, at three and/or six weeks of age, littermates were placed in a clean cage and underwent 30 minute video-taped observations between 1:00 and 5:00pm. Prior extensive video-electroencephalogram monitoring of Q54 mice demonstrated a strong correlation between behavioral and electroencephalographic seizures (κ = 0.988), validating visual assessment of seizures.1; 11 Behavioral seizures were assessed offline by an observer blinded to genotype, counting the number of focal motor seizures (FMS) with forelimb clonus and repetitive movements lasting one to five seconds. For evaluation of transgenic lines SJL.1GL and B6.1GH, seizure counts from three and six weeks were combined to obtain a 60-minute seizure frequency for each animal. For F1.Q54;1GKO/+ evaluation, seizure counts from the three week time-point were used for analysis. Other seizure types, including generalized tonic-clonic seizures (GTCS), were rarely observed at these ages. Seizure frequencies were compared between genotypes using Student’s T-test (parametric) or Mann-Whitney rank-sum test (nonparametric).
Results and Discussion Author Manuscript
We previously mapped Moe1, a dominant modifier locus on chromosome 11 that influenced Scn2aQ54 seizure frequency. Based on fine-mapping and RNA-Seq analysis, we nominated Cacna1g as a candidate modifier gene at Moe1.4 To evaluate the modifier effect of Cacna1g on the Scn2aQ54 seizure phenotype, we generated two independent Cacna1g BAC transgenic mouse lines: SJL.Cacna1gLOW (SJL.1GL) and B6.Cacna1gHIGH (B6.1GH), with low and high transgene expression, respectively. During routine breeding of SJL.1GL and B6.1GH
Epilepsia. Author manuscript; available in PMC 2017 June 01.
Calhoun et al.
Page 5
Author Manuscript
lines, X-linked inheritance patterns became apparent. Due to the potential for X-inactivation of the BAC transgenes as a confounding factor, we focused our efforts on studying males. We confirmed integrity of the 1GL and 1GH BAC constructs using vector primers and microsatellite markers spanning the BAC. Copy number analysis estimated 5–8 transgene copy integrations for both SJL.1GL and B6.1GH lines. A modest increase in Cacna1g transcript expression was observed in hemizygous SJL.1GL compared to wildtype littermates (p
Epilepsia. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Epilepsia. 2016 June ; 57(6): e103–e107. doi:10.1111/epi.13390.
Cacna1g is a genetic modifier of epilepsy caused by mutation of voltage-gated sodium channel Scn2a Jeffrey D. Calhoun1,2,*, Nicole A. Hawkins1,3,*, Nicole J. Zachwieja1, and Jennifer A. Kearney1,2,† 1Department
of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, 60611, USA
Author Manuscript
2Department
of Medicine, Vanderbilt University, Nashville, Tennessee, 37232, USA
3Neuroscience
Program, Vanderbilt University, Nashville, Tennessee, 37232, USA
Summary
Author Manuscript
More than 1200 mutations in neuronal voltage-gated sodium channel genes have been identified in patients with several epilepsy syndromes. A common feature of genetic epilepsies is variable expressivity among individuals with the same mutation. The Scn2aQ54 transgenic mouse model has a mutation in Scn2a that results in spontaneous epilepsy. Scn2aQ54 phenotype severity varies depending on the genetic strain background, making it a useful model for identifying and characterizing epilepsy modifier genes. Scn2aQ54 mice on the [C57BL/6JxSJL/J]F1 background exhibit earlier seizure onset, elevated spontaneous seizure frequency, and decreased survival compared to Scn2aQ54 mice congenic on the C57BL/6J strain. Genetic mapping and RNA-Seq analysis identified Cacna1g as a candidate modifier gene at the Moe1 locus, which influences Scn2aQ54 phenotype severity. In this study, we evaluated the modifier potential of Cacna1g, encoding the Cav3.1 voltage-gated calcium channel, by testing whether transgenic alteration of Cacna1g expression modifies severity of the Scn2aQ54 seizure phenotype. Scn2aQ54 mice exhibited increased spontaneous seizure frequency with elevated Cacna1g expression and decreased seizure frequency with decreased Cacna1g expression. These results provide support for Cacna1g as an epilepsy modifier gene and suggest that modulation of Cav3.1 may be an effective therapeutic strategy.
Keywords
Author Manuscript
Seizures; Mouse model; Voltage-gated ion channels; Voltage-gated calcium channels; Genetics
†
Corresponding Author Jennifer A. Kearney, Department of Pharmacology, Searle 8-510, 320 East Superior St. Northwestern University, Chicago, IL 60611, Tel: 312-503-4894, [email protected]. *indicates equal contribution Disclosure None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
Calhoun et al.
Page 2
Author Manuscript
Introduction Mutations in the voltage-gated sodium channel (VGSC) genes SCN1A, SCN2A, SCN3A, and SCN8A are responsible for several human epilepsies. More than 1200 mutations in SCN1A, SCN2A, and SCN8A have been identified in patients with various epilepsy syndromes, including Genetic Epilepsy with Febrile Seizures Plus, Benign Familial Neonatal-Infantile Seizures, and early infantile epileptic encephalopathies. Variable expressivity is a common feature of epilepsies caused by VGSC mutations.
Author Manuscript
Mouse epilepsy models provide a tractable system to map and characterize modifier genes. A common feature of these models is strain-dependent phenotype severity, suggesting the contribution of genetic modifiers. Scn2aQ54 (Q54) transgenic mice have a gain-of-function mutation that results in spontaneous focal motor seizures and strain-dependent epilepsy severity.1; 2 On the C57BL/6J (B6) strain, the Q54 mutation (B6.Q54) results in mild, adultonset epilepsy. When B6.Q54 is crossed to SJL/J (SJL), the resulting F1.Q54 mice experience juvenile-onset spontaneous seizures and reduced lifespan. We previously mapped modifier loci responsible for the strain difference in seizure frequency exhibited by Scn2aQ54 mice: Moe1 (modifier of epilepsy 1) and Moe2 on chromosome 11 and 19, respectively.2 Kcnv2 was identified as a modifier gene at Moe2.3 Recent work indicated multiple genes likely underlie the Moe1 peak, including Cacna1g and Hlf.4 Genetic abalation of Hlf, encoding a PAR bZIP transcription factor, exacerbated epilepsy in the Scn2aQ54 and Scn1a+/− Dravet mouse models.5
Author Manuscript
Cacna1g resides within the Moe1 locus and RNA-seq showed strain-dependent differences in Cacna1g transcript expression and alternative splicing.4 Cacna1g encodes the poreforming α1G/Cav3.1 subunit, a low-voltage activated calcium channel of the T-type calcium channel family. Patient and animal model data have demonstrated a link between T-type currents and absence seizures. Ethosuximide (ETX), a T-type channel blocker, is widely used for treatment of absence epilepsy in patients. In mice, targeted deletion of Cacna1g diminishes GABAB-receptor agonist-induced spike-wave discharges (SWDs), while overexpression of Cacna1g is associated with absence seizures and abnormal thalamocortical synchronization.6–8 A role for Cacna1g in non-absence epilepsy has been remained elusive. This study evaluated the modifier effect of Cacna1g on the Scn2aQ54 mouse epilepsy model. We tested the effect of transgenic upregulation or heterozygous deletion of Cacna1g on spontaneous seizure frequency of Scn2aQ54 mice.
Author Manuscript
Methods Mice A B6-derived BAC clone containing Cacna1g (RP23-65I14) was obtained from BACPAC Resources (http://bacpac.chori.org/home.htm). Pronuclear injections into fertilized SJL or B6 oocytes were performed by the Vanderbilt University Transgenic Mouse/ESC Shared Resource and the University of Michigan Transgenic Animal Model Core, respectively. Injections resulted in two Cacna1g BAC transgenic founders: (1) SJL.Cacna1gLOW (SJL.
Epilepsia. Author manuscript; available in PMC 2017 June 01.
Calhoun et al.
Page 3
Author Manuscript
1GL), maintained by continued backcrossing of hemizygous transgenic mice to SJL/J (Jackson Labs); and (2) B6.Cacna1gHIGH (B6.1GH), maintained by continued backcrossing of hemizygous transgenic mice to C57BL/6J (Jackson Labs). B6(129S4)Cacna1gtm1Stl/J mice were obtained (Jackson Labs #021932), bred to C57BL/6-Tg(Zp3cre)93Knw/J (Jackson Labs #003651), and maintained by continued backcrossing of heterozygous Cacna1gKO/+ (B6.1GKO/+) offspring to B6.Scn2aQ54 transgenic mice [Tg(Eno2-Scn2a1*)Q54Mm] congenic on B6 (B6.Q54) were maintained by continued backcrossing of hemizygous B6.Q54 males to B6 females.1
Author Manuscript
Transgenic SJL.1GL (SJL/J background) and B6.1GH (C57BL/6J background) females were crossed with B6.Q54 males to generate double transgenic (F1.Q54;1GL or B6.Q54;1GH) mice and single transgenic littermate controls. B6.1GKO/+ females were crossed to B6.Q54 mice, and B6.Q54;1GKO/+ male offspring were subsequently crossed to SJL/J females to generate F1.Q54;1GKO/+ mice and single transgenic littermate controls. Mice were group-housed with access to food and water ad libitum. All studies were approved by the Vanderbilt University and Northwestern University Animal Care and Use Committees in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The principles outlined in the ARRIVE guideline and Basel declaration (including the 3R concept) were considered when planning the experiments. Genotyping
Author Manuscript
Mice were genotyped by PCR of DNA isolated from postnatal day 14 (P14) tail biopsies. SJL.1GL and B6.1GH transgenic mice were identified using 1G-SP6 and 1G-T7 primers that amplify 200 bp and 220 bp products, respectively. The Q54 transgene and 1GKO/+ allele were genotyped as described.1; 9 Primer sequences used for genotyping, BAC integrity, copy number, and expression analysis will be provided upon request. BAC Integrity and Copy Number BAC integrity was evaluated by PCR amplification of genomic DNA. SP6 and T7 promoters flanked each end of the BAC insert, while microsatellite markers were located approximately every 32 Kb, including two within Cacna1g. B6.1GH mice were backcrossed twice to SJL to obtain informative mice for integrity analysis of the B6-derived BAC.
Author Manuscript
Transgene copy number was assessed by quantitative PCR (qPCR) using Cacna1g and reference Scn5a Taqman assays previously described.8; 10 At least n>4 independent DNA samples from hemizygous transgenics and wildtype controls were used to estimate copy number. Standard curves were generated by spiking SJL or B6 genomic DNA with BAC plasmid DNA equivalent to 0, 5, 10 and 20 copies per diploid genome. Transcript Analysis and Protein Analysis RNA was extracted from whole brains of six week old mice using Trizol (Life Technologies). The six week time point was selected to match our previous RNA-seq transcriptome analysis that identified Cacna1g as a candidate modifier.4 Total RNA was reverse transcribed using SuperScript III (Life Technologies). Quantitative digital droplet
Epilepsia. Author manuscript; available in PMC 2017 June 01.
Calhoun et al.
Page 4
Author Manuscript
PCR (ddPCR) was performed using ddPCR Supermix for Probes (No dUTP) (Bio-Rad) and TaqMan Gene Expression Assays (Life Technologies) for Cacna1g (FAMMm00486571_m1) and TATA binding protein (Tbp) (VIC-Mm00446971_m1). Reactions were partitioned into 20,000 droplets (1 nl each) in a QX200 droplet generator (Bio-Rad). Thermocycling conditions were 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute (ramp rate of 2°C/sec), and a final inactivation step of 98°C for 10 minutes. Following amplification, droplets were analyzed with a QX200 droplet reader and QuantaSoft v1.6.6.0320 software (Bio-Rad). All assays lacked detectable signal in no-RT and no-template controls. Relative transcript levels were expressed as a ratio of Cacna1g/Tbp concentration and compared between groups by Student’s T-test.
Author Manuscript
Membrane protein fractions (50 μg) were prepared from whole brains of six week old mice and separated on 4–20% gradient SDS-PAGE gels (Biorad) and transferred to nitrocellulose. Membranes were assayed using mouse monoclonal Cav3.1 (Neuromab, N178A/9; 1:500) and β-tubulin (Sigma, TUB 2.1; 1:2,000) primary antibodies, and peroxidase-conjugated goat anti-mouse (1:50,000; Jackson ImmunoResearch) secondary antibodies. Signals were detected with SuperSignal West Femto (Pierce) and densitometry was performed using ImageJ. Cav3.1 expression was normalized to the corresponding β-tubulin band. Expression differences were compared between groups by Student’s T-test. Phenotyping
Author Manuscript
Transgenic mice were phenotyped as previously described.4 Briefly, at three and/or six weeks of age, littermates were placed in a clean cage and underwent 30 minute video-taped observations between 1:00 and 5:00pm. Prior extensive video-electroencephalogram monitoring of Q54 mice demonstrated a strong correlation between behavioral and electroencephalographic seizures (κ = 0.988), validating visual assessment of seizures.1; 11 Behavioral seizures were assessed offline by an observer blinded to genotype, counting the number of focal motor seizures (FMS) with forelimb clonus and repetitive movements lasting one to five seconds. For evaluation of transgenic lines SJL.1GL and B6.1GH, seizure counts from three and six weeks were combined to obtain a 60-minute seizure frequency for each animal. For F1.Q54;1GKO/+ evaluation, seizure counts from the three week time-point were used for analysis. Other seizure types, including generalized tonic-clonic seizures (GTCS), were rarely observed at these ages. Seizure frequencies were compared between genotypes using Student’s T-test (parametric) or Mann-Whitney rank-sum test (nonparametric).
Results and Discussion Author Manuscript
We previously mapped Moe1, a dominant modifier locus on chromosome 11 that influenced Scn2aQ54 seizure frequency. Based on fine-mapping and RNA-Seq analysis, we nominated Cacna1g as a candidate modifier gene at Moe1.4 To evaluate the modifier effect of Cacna1g on the Scn2aQ54 seizure phenotype, we generated two independent Cacna1g BAC transgenic mouse lines: SJL.Cacna1gLOW (SJL.1GL) and B6.Cacna1gHIGH (B6.1GH), with low and high transgene expression, respectively. During routine breeding of SJL.1GL and B6.1GH
Epilepsia. Author manuscript; available in PMC 2017 June 01.
Calhoun et al.
Page 5
Author Manuscript
lines, X-linked inheritance patterns became apparent. Due to the potential for X-inactivation of the BAC transgenes as a confounding factor, we focused our efforts on studying males. We confirmed integrity of the 1GL and 1GH BAC constructs using vector primers and microsatellite markers spanning the BAC. Copy number analysis estimated 5–8 transgene copy integrations for both SJL.1GL and B6.1GH lines. A modest increase in Cacna1g transcript expression was observed in hemizygous SJL.1GL compared to wildtype littermates (p
