https://www.alphaknockout.com
Mouse Glra2 Knockout Project (CRISPR/Cas9)
Objective: To create a Glra2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Glra2 gene (NCBI Reference Sequence: NM_183427 ; Ensembl: ENSMUSG00000018589 ) is located on Mouse chromosome X. 9 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000058787). Exon 3~4 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a null allele lack cortical neuron responses to glycine and taurine but are otherwise normal. Mice homozygous for another targeted allele exhibit impaired interneuron migration into the cortical wall.
Exon 3 starts from about 14.97% of the coding region. Exon 3~4 covers 21.53% of the coding region. The size of effective KO region: ~7844 bp. The KO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele 5' gRNA region gRNA region 3'
1 3 4 9
Legends Exon of mouse Glra2 Knockout region
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Overview of the Dot Plot (up) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 2000 bp section upstream of Exon 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.
Overview of the Dot Plot (down) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 2000 bp section downstream of Exon 4 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.
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Overview of the GC Content Distribution (up) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(32.45% 649) | C(16.95% 339) | T(32.05% 641) | G(18.55% 371)
Note: The 2000 bp section upstream of Exon 3 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.
Overview of the GC Content Distribution (down) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(25.3% 506) | C(19.25% 385) | T(31.7% 634) | G(23.75% 475)
Note: The 2000 bp section downstream of Exon 4 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 2000 1 2000 2000 100.0% chrX - 165289667 165291666 2000 browser details YourSeq 45 145 321 2000 94.2% chr11 + 108378545 108378726 182 browser details YourSeq 40 86 136 2000 95.6% chr11 - 113023475 113023720 246 browser details YourSeq 32 189 294 2000 94.5% chr2 - 75551625 75551730 106 browser details YourSeq 32 203 354 2000 91.5% chr17 + 56991736 56991886 151 browser details YourSeq 30 1113 1154 2000 97.1% chr1 + 119811126 119811170 45 browser details YourSeq 29 287 342 2000 72.1% chr5 + 98443396 98443446 51 browser details YourSeq 29 313 347 2000 87.9% chr10 + 51164641 51164674 34 browser details YourSeq 25 282 324 2000 79.1% chr4 + 41430115 41430157 43 browser details YourSeq 24 125 154 2000 84.7% chr13 - 109635825 109635852 28 browser details YourSeq 20 194 215 2000 95.5% chr12 + 12597016 12597037 22
Note: The 2000 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 2000 1 2000 2000 100.0% chrX - 165279823 165281822 2000 browser details YourSeq 589 791 1739 2000 87.8% chr15 - 79838205 79839156 952 browser details YourSeq 589 788 1739 2000 86.3% chr4 + 12396151 12397097 947 browser details YourSeq 564 807 1739 2000 88.7% chr2 + 18448628 18449567 940 browser details YourSeq 563 807 1739 2000 87.5% chr11 + 68752772 68753709 938 browser details YourSeq 561 814 1731 2000 87.1% chr18 + 5911906 5912845 940 browser details YourSeq 556 807 1739 2000 88.6% chr5 + 123982754 123983679 926 browser details YourSeq 554 811 1736 2000 87.7% chr7 - 129587890 129588795 906 browser details YourSeq 552 807 1739 2000 86.8% chr5 - 22151287 22152218 932 browser details YourSeq 550 806 1739 2000 86.0% chr13 + 58371792 58372735 944 browser details YourSeq 546 817 1747 2000 86.2% chr11 - 48062700 48064050 1351 browser details YourSeq 541 910 1739 2000 86.7% chr15 + 79848436 79849234 799 browser details YourSeq 540 792 1739 2000 84.9% chr5 - 48839103 48840062 960 browser details YourSeq 540 811 1739 2000 83.3% chr12 - 112079479 112080399 921 browser details YourSeq 537 788 1739 2000 85.7% chr10 + 127861351 127862255 905 browser details YourSeq 535 811 1740 2000 87.0% chr19 + 61283506 61284396 891 browser details YourSeq 531 807 1739 2000 85.3% chr5 - 86502167 86503066 900 browser details YourSeq 531 787 1739 2000 86.6% chr19 + 14688025 14688901 877 browser details YourSeq 526 815 1739 2000 87.3% chr4 - 80136637 80137574 938 browser details YourSeq 525 819 1689 2000 84.6% chr1 + 173443644 173444478 835
Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Glra2 glycine receptor, alpha 2 subunit [ Mus musculus (house mouse) ] Gene ID: 237213, updated on 24-Oct-2019
Gene summary
Official Symbol Glra2 provided by MGI Official Full Name glycine receptor, alpha 2 subunit provided by MGI Primary source MGI:MGI:95748 See related Ensembl:ENSMUSG00000018589 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 Expression Biased expression in CNS E18 (RPKM 14.3), whole brain E14.5 (RPKM 8.6) and 4 other tissues See more Orthologs human all
Genomic context
Location: X F5; X 76.75 cM See Glra2 in Genome Data Viewer
Exon count: 12
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (165129017..165327867, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (161566949..161764913, complement)
Chromosome X - NC_000086.7
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Transcript information: This gene has 1 transcript
Gene: Glra2 ENSMUSG00000018589
Description glycine receptor, alpha 2 subunit [Source:MGI Symbol;Acc:MGI:95748] Location Chromosome X: 165,129,017-165,327,393 reverse strand. GRCm38:CM001013.2 About this gene This gene has 1 transcript (splice variant), 191 orthologues, 41 paralogues, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Glra2-201 ENSMUST00000058787.8 3150 452aa ENSMUSP00000060827.8 Protein coding CCDS41206 Q3UTL8 Q7TNC8 TSL:1 GENCODE basic APPRIS P1
218.38 kb Forward strand 165.15Mb 165.20Mb 165.25Mb 165.30Mb Contigs AL672152.8 > AL831734.10 > Genes (Comprehensive set... < Glra2-201protein coding
Regulatory Build
165.15Mb 165.20Mb 165.25Mb 165.30Mb Reverse strand 218.38 kb
Regulation Legend CTCF Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
merged Ensembl/Havana
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Transcript: ENSMUST00000058787
< Glra2-201protein coding
Reverse strand 198.38 kb
ENSMUSP00000060... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... TIGRFAM Neurotransmitter-gated ion-channel Superfamily Neurotransmitter-gated ion-channel ligand-binding domain superfamily
Neurotransmitter-gated ion-channel transmembrane domain superfamily Prints Glycine receptor alpha
Gamma-aminobutyric acid A receptor/Glycine receptor alpha
Neurotransmitter-gated ion-channel
Glycine receptor alpha2 Pfam Neurotransmitter-gated ion-channel ligand-binding domain Neurotransmitter-gated ion-channel transmembrane domain
PROSITE patterns Neurotransmitter-gated ion-channel, conserved site PANTHER Neurotransmitter-gated ion-channel
Glycine receptor alpha2 Gene3D Neurotransmitter-gated ion-channel ligand-binding domain superfamily CDD cd19009 cd19060
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant synonymous variant
Scale bar 0 40 80 120 160 200 240 280 320 360 400 452
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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