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Mouse P2rx6 Conditional Knockout Project (CRISPR/Cas9)

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Mouse P2rx6 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse P2rx6 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a P2rx6 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The P2rx6 gene (NCBI Reference Sequence: NM_011028 ; Ensembl: ENSMUSG00000022758 ) is located on Mouse chromosome 16. 12 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 12 (Transcript: ENSMUST00000023441). Exon 4~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse P2rx6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-180P3 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: Homozygous mutant mice exhibit a significant increase in thermal response latency during hot plate testing, and are resistant to metrazol-induced seizures. Exon 4 starts from about 33.76% of the coding region. The knockout of Exon 4~8 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2410 bp, and the size of intron 8 for 3'-loxP site insertion: 1987 bp. The size of effective cKO region: ~1433 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 4 5 6 7 8 9 10 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse P2rx6 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com 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(7933bp) | A(23.36% 1853) | C(26.66% 2115) | T(26.09% 2070) | G(23.89% 1895) 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. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr16 + 17564189 17567188 3000 browser details YourSeq 40 334 377 3000 97.7% chr1 - 90899611 90899656 46 browser details YourSeq 36 131 180 3000 81.7% chr8 - 36189541 36189589 49 browser details YourSeq 35 131 182 3000 86.1% chr11 - 22550671 22550721 51 browser details YourSeq 33 317 408 3000 89.2% chr8 + 16246696 16246786 91 browser details YourSeq 27 2523 2552 3000 96.7% chr1 + 35871178 35871208 31 browser details YourSeq 26 131 158 3000 96.5% chr8 - 127112511 127112538 28 browser details YourSeq 20 384 403 3000 100.0% chr8 - 40875169 40875188 20 browser details YourSeq 20 384 403 3000 100.0% chr4 + 16214235 16214254 20 Note: The 3000 bp section upstream of Exon 4 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% chr16 + 17568622 17571621 3000 browser details YourSeq 279 12 641 3000 87.4% chr2 - 92464998 92959496 494499 browser details YourSeq 245 15 532 3000 88.0% chr11 - 6800792 6801362 571 browser details YourSeq 233 3 639 3000 86.6% chr16 - 13614261 13614945 685 browser details YourSeq 217 4 453 3000 86.3% chr9 + 22252904 22253411 508 browser details YourSeq 201 291 641 3000 88.4% chrX + 12530264 12530614 351 browser details YourSeq 192 330 866 3000 83.6% chr11 + 31818482 31818974 493 browser details YourSeq 191 21 532 3000 89.1% chrX - 152285807 152286319 513 browser details YourSeq 176 293 617 3000 87.5% chr3 + 89699238 89699582 345 browser details YourSeq 176 283 641 3000 87.8% chr13 + 32412362 32412733 372 browser details YourSeq 171 283 641 3000 83.5% chr12 - 112832477 112832833 357 browser details YourSeq 171 283 622 3000 83.3% chr11 - 106508863 106509172 310 browser details YourSeq 170 312 617 3000 89.1% chr9 - 73788689 73789011 323 browser details YourSeq 167 293 619 3000 86.2% chr18 - 64507016 64507345 330 browser details YourSeq 165 292 641 3000 90.4% chr15 + 38188806 38189178 373 browser details YourSeq 163 311 622 3000 88.1% chr16 + 30245605 30245927 323 browser details YourSeq 161 387 645 3000 86.4% chr10 - 68340189 68340468 280 browser details YourSeq 159 328 579 3000 87.0% chr7 - 99294477 99294733 257 browser details YourSeq 157 283 620 3000 84.2% chr11 - 97917441 97917773 333 browser details YourSeq 156 358 617 3000 86.5% chr13 - 30105527 30105785 259 Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: P2rx6 purinergic receptor P2X, ligand-gated ion channel, 6 [ Mus musculus (house mouse) ] Gene ID: 18440, updated on 10-Oct-2019 Gene summary Official Symbol P2rx6 provided by MGI Official Full Name purinergic receptor P2X, ligand-gated ion channel, 6 provided by MGI Primary source MGI:MGI:1337113 See related Ensembl:ENSMUSG00000022758 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 P2x6; P2xm; P2rxl1 Expression Broad expression in subcutaneous fat pad adult (RPKM 7.9), ovary adult (RPKM 5.8) and 20 other tissues See more Orthologs human all Genomic context Location: 16; 16 A3 See P2rx6 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (17561867..17572015) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (17561978..17572105) Chromosome 16 - NC_000082.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: P2rx6 ENSMUSG00000022758 Description purinergic receptor P2X, ligand-gated ion channel, 6 [Source:MGI Symbol;Acc:MGI:1337113] Gene Synonyms P2rxl1, P2xm Location Chromosome 16: 17,561,885-17,577,800 forward strand. GRCm38:CM001009.2 About this gene This gene has 3 transcripts (splice variants), 122 orthologues, 6 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags P2rx6- ENSMUST00000023441.10 2345 389aa ENSMUSP00000023441.4 Protein coding CCDS28007 O54803 TSL:1 201 GENCODE basic APPRIS P1 P2rx6- ENSMUST00000171002.9 1353 362aa ENSMUSP00000132727.1 Protein coding CCDS49778 E9PY29 TSL:1 202 GENCODE basic P2rx6- ENSMUST00000231806.1 3382 286aa ENSMUSP00000156201.1 Nonsense mediated - A0A338P727 - 203 decay Page 6 of 8 https://www.alphaknockout.com 35.92 kb Forward strand 17.56Mb 17.57Mb 17.58Mb Genes (Comprehensive set... P2rx6-201 >protein coding P2rx6-202 >protein coding P2rx6-203 >nonsense mediated decay Contigs AC115733.14 > Genes < Lrrc74b-201protein coding < Slc7a4-202protein coding (Comprehensive set... < Lrrc74b-202protein coding < Slc7a4-201protein coding < Lrrc74b-203lncRNA < Slc7a4-205protein coding < Lrrc74b-204protein coding < Slc7a4-210protein coding < Slc7a4-211retained intron < Slc7a4-208protein coding < Slc7a4-206protein coding < Slc7a4-204protein coding < Slc7a4-203protein coding < Slc7a4-209protein coding < Slc7a4-207protein coding Regulatory Build 17.56Mb 17.57Mb 17.58Mb Reverse strand 35.92 kb Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000023441 10.13 kb Forward strand P2rx6-201 >protein coding ENSMUSP00000023... Transmembrane heli... Low complexity (Seg) TIGRFAM P2X purinoreceptor Prints P2X6 purinoceptor P2X purinoreceptor Pfam PF00864 PROSITE patterns P2X purinoreceptor PIRSF P2X purinoreceptor PANTHER P2X6 purinoceptor PTHR10125 Gene3D 1.10.287.940 P2X purinoreceptor extracellular domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 389 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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