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Mouse Cacna1b Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Cacna1b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Cacna1b gene (NCBI Reference Sequence: NM_001042528 ; Ensembl: ENSMUSG00000004113 ) is located on Mouse chromosome 2. 47 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 47 (Transcript: ENSMUST00000041342). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cacna1b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-474E19 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice deficient in this gene exhibit defects in nociception, memory and learning. They also exhibit hyperactive and hyperaggressive behaviors as well as defects in the the sleep-wake cycle. Deficits in the sympathetic nervous system results in defects in circulatory regulation.
Exon 3 starts from about 5.6% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 3354 bp, and the size of intron 3 for 3'-loxP site insertion: 24245 bp. The size of effective cKO region: ~640 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 3 47 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Cacna1b Homology arm cKO region loxP site
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Overview of the Dot Plot Window size: 10 bp
Forward Reverse Complement
Sequence 12
Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.
Overview of the GC Content Distribution Window size: 300 bp
Sequence 12
Summary: Full Length(7140bp) | A(20.63% 1473) | C(24.58% 1755) | T(27.65% 1974) | G(27.14% 1938)
Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.
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BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr2 - 24758672 24761671 3000 browser details YourSeq 166 652 847 3000 94.2% chr1 + 161140716 161140920 205 browser details YourSeq 153 652 840 3000 92.7% chr4 + 149447915 149448103 189 browser details YourSeq 152 652 945 3000 93.7% chr2 - 125262593 125262922 330 browser details YourSeq 152 656 846 3000 89.2% chr1 - 54586597 54586763 167 browser details YourSeq 150 656 836 3000 92.8% chr18 - 34278327 34278513 187 browser details YourSeq 148 656 842 3000 88.7% chr13 + 9007570 9007739 170 browser details YourSeq 146 650 835 3000 87.8% chr5 - 3597811 3597983 173 browser details YourSeq 146 652 844 3000 88.2% chr14 + 73016470 73016629 160 browser details YourSeq 145 661 839 3000 92.8% chr16 - 68573219 68573396 178 browser details YourSeq 145 657 845 3000 87.3% chr4 + 133819459 133819623 165 browser details YourSeq 143 662 842 3000 89.0% chr15 - 5889638 5889805 168 browser details YourSeq 143 662 842 3000 88.2% chr5 + 117496156 117496318 163 browser details YourSeq 142 653 845 3000 87.4% chr10 - 128910949 128911106 158 browser details YourSeq 142 656 845 3000 85.4% chrX + 135573471 135573642 172 browser details YourSeq 142 651 842 3000 89.1% chr2 + 142670970 142671151 182 browser details YourSeq 142 651 846 3000 91.3% chr18 + 18903468 18903666 199 browser details YourSeq 142 652 841 3000 88.7% chr12 + 36097560 36097739 180 browser details YourSeq 141 641 842 3000 88.8% chr12 - 3603741 3603938 198 browser details YourSeq 139 652 806 3000 92.9% chr4 - 54482231 54482383 153
Note: The 3000 bp section upstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.
BLAT Search Results (down)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr2 - 24755032 24758031 3000 browser details YourSeq 177 271 580 3000 93.7% chr3 + 153946353 153946700 348 browser details YourSeq 176 262 585 3000 93.2% chr1 + 136387916 136388508 593 browser details YourSeq 160 261 589 3000 87.9% chr15 + 36411314 36411635 322 browser details YourSeq 154 266 585 3000 88.2% chr7 + 79389486 79389972 487 browser details YourSeq 151 215 421 3000 92.6% chr13 + 44853153 44853462 310 browser details YourSeq 146 257 422 3000 95.1% chr7 - 30116809 30116977 169 browser details YourSeq 146 262 424 3000 95.1% chr12 - 97190549 97190713 165 browser details YourSeq 145 257 421 3000 93.9% chr2 - 69246643 69246806 164 browser details YourSeq 145 264 480 3000 91.5% chr10 - 43894415 43894987 573 browser details YourSeq 144 261 423 3000 95.1% chr6 + 29297409 29297571 163 browser details YourSeq 144 273 580 3000 94.0% chr3 + 9275494 9275819 326 browser details YourSeq 144 257 425 3000 91.6% chr2 + 9851599 9851765 167 browser details YourSeq 144 261 422 3000 93.2% chr13 + 73638166 73638326 161 browser details YourSeq 143 262 421 3000 95.0% chr5 - 110574323 110574483 161 browser details YourSeq 143 260 423 3000 95.0% chr14 - 59955426 59955591 166 browser details YourSeq 143 261 418 3000 96.2% chr15 + 70865017 70865182 166 browser details YourSeq 143 260 425 3000 90.7% chr1 + 18256357 18256518 162 browser details YourSeq 142 261 422 3000 93.2% chrX + 57776383 57776543 161 browser details YourSeq 142 260 428 3000 92.9% chr7 + 139934755 139934930 176
Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Cacna1b calcium channel, voltage-dependent, N type, alpha 1B subunit [ Mus musculus (house mouse) ] Gene ID: 12287, updated on 12-Aug-2019
Gene summary
Official Symbol Cacna1b provided by MGI Official Full Name calcium channel, voltage-dependent, N type, alpha 1B subunit provided by MGI Primary source MGI:MGI:88296 See related Ensembl:ENSMUSG00000004113 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as BIII; Cav2.2; Cchn1a; AW050276; AW060892; AW822256; alpha(1B) Expression Biased expression in CNS E18 (RPKM 11.3), whole brain E14.5 (RPKM 9.4) and 8 other tissues See more Orthologs human all
Genomic context
Location: 2 A3; 2 16.58 cM See Cacna1b in Genome Data Viewer
Exon count: 52
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (24603860..24763200, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (24461895..24618672, complement)
Chromosome 2 - NC_000068.7
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Transcript information: This gene has 10 transcripts
Gene: Cacna1b ENSMUSG00000004113
Description calcium channel, voltage-dependent, N type, alpha 1B subunit [Source:MGI Symbol;Acc:MGI:88296] Gene Synonyms Cav2.2, Cchn1a, alpha(1B) Location Chromosome 2: 24,603,887-24,763,152 reverse strand. GRCm38:CM000995.2 About this gene This gene has 10 transcripts (splice variants), 269 orthologues, 26 paralogues, is a member of 1 Ensembl protein family and is associated with 62 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Cacna1b- ENSMUST00000070864.13 9655 2288aa ENSMUSP00000063236.7 Protein coding CCDS15739 A2AIS0 TSL:5 202 GENCODE basic APPRIS P3
Cacna1b- ENSMUST00000041342.11 6984 2327aa ENSMUSP00000037416.5 Protein coding CCDS38065 O55017 TSL:1 201 GENCODE basic APPRIS ALT2
Cacna1b- ENSMUST00000102939.8 9736 2325aa ENSMUSP00000100003.2 Protein coding - A2AIR9 TSL:5 204 GENCODE basic APPRIS ALT2
Cacna1b- ENSMUST00000100348.9 7182 2328aa ENSMUSP00000097920.3 Protein coding - A2AIR7 TSL:5 203 GENCODE basic APPRIS ALT2
Cacna1b- ENSMUST00000114447.7 6987 2328aa ENSMUSP00000110090.1 Protein coding - A2AIR7 TSL:5 205 GENCODE basic APPRIS ALT2
Cacna1b- ENSMUST00000133892.1 684 228aa ENSMUSP00000115285.1 Protein coding - A2AIZ9 CDS 5' and 3' 209 incomplete TSL:3
Cacna1b- ENSMUST00000131861.1 523 168aa ENSMUSP00000141653.1 Protein coding - A0A0A6YWR0 CDS 5' 208 incomplete TSL:5
Cacna1b- ENSMUST00000155356.3 332 111aa ENSMUSP00000116674.2 Protein coding - F7C9S8 CDS 5' and 3' 210 incomplete TSL:5
Cacna1b- ENSMUST00000124183.1 308 102aa ENSMUSP00000114605.1 Protein coding - A2AJ01 CDS 5' and 3' 206 incomplete TSL:5
Cacna1b- ENSMUST00000125798.2 949 114aa ENSMUSP00000141767.1 Nonsense mediated - A0A0A6YWZ5 CDS 5' 207 decay incomplete TSL:5
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179.27 kb Forward strand
24.60Mb 24.65Mb 24.70Mb 24.75Mb Genes Gm25455-201 >misc RNA Gm13459-201 >processed pseudogene (Comprehensive set...
Contigs AL732546.13 > BX294112.6 > AL732525.13 > Genes (Comprehensive set... < Gm13416-201processed pseudogene < Cacna1b-206protein coding
< Cacna1b-202protein coding
< Cacna1b-204protein coding
< Cacna1b-203protein coding
< Cacna1b-208protein coding
< Cacna1b-205protein coding
< Cacna1b-201protein coding
< Cacna1b-209protein coding
< Cacna1b-207nonsense mediated decay
< Cacna1b-210protein coding
Regulatory Build
24.60Mb 24.65Mb 24.70Mb 24.75Mb Reverse strand 179.27 kb
Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site
Gene Legend Protein Coding
merged Ensembl/Havana Ensembl protein coding
Non-Protein Coding
pseudogene processed transcript RNA gene
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Transcript: ENSMUST00000041342
< Cacna1b-201protein coding
Reverse strand 156.48 kb
ENSMUSP00000037... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF81324 SMART Voltage-dependent calcium channel, alpha-1 subunit, IQ domain
Prints Voltage-dependent calcium channel, N-type, alpha-1 subunit
Voltage-dependent calcium channel, alpha-1 subunit Pfam Ion transport domain Voltage-dependent L-type calcium channel, IQ-associated domain
Voltage-dependent calcium channel, alpha-1 subunit, IQ domain PROSITE profiles EF-hand domain
PANTHER PTHR45628
Voltage-dependent calcium channel, N-type, alpha-1 subunit Gene3D Voltage-dependent channel domain superfamily 1.10.238.10
1.10.287.70
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend inframe insertion missense variant splice region variant synonymous variant
Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2327
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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