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Mouse Pag1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Pag1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pag1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pag1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pag1 gene (NCBI Reference Sequence: NM_001195031 ; Ensembl: ENSMUSG00000027508 ) is located on Mouse chromosome 3. 9 exons are identified, with the ATG start codon in exon 4 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000161949). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pag1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-97H4 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 homozygous for a knock-out allele are viable and exhibit no apparent defects in embryogenesis, thymic development, or T-cell functions. Mice homozygous for a different knock-out allele show normal T-cell development albeit with an increased thymocyte population. Exon 5 starts from about 10.26% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1327 bp, and the size of intron 5 for 3'-loxP site insertion: 2649 bp. The size of effective cKO region: ~552 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 5 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pag1 Homology arm cKO region loxP site Page 2 of 7 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(7052bp) | A(23.36% 1647) | C(25.24% 1780) | T(26.53% 1871) | G(24.87% 1754) 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 7 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% chr3 - 9705006 9708005 3000 browser details YourSeq 33 969 1015 3000 94.8% chr4 - 99257799 99257846 48 browser details YourSeq 28 1054 1095 3000 96.7% chr8 - 107317829 107317870 42 browser details YourSeq 26 1040 1068 3000 85.2% chr2 + 69697886 69697912 27 Note: The 3000 bp section upstream of Exon 5 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% chr3 - 9701454 9704453 3000 browser details YourSeq 43 2580 2687 3000 71.8% chr4 - 55597387 55597445 59 browser details YourSeq 39 2573 2612 3000 100.0% chr11 + 108963290 108963333 44 browser details YourSeq 36 2569 2611 3000 93.1% chr10 + 24779246 24779293 48 browser details YourSeq 35 2573 2608 3000 100.0% chr4 - 133349432 133349471 40 browser details YourSeq 34 2573 2609 3000 97.3% chr2 + 121772566 121772606 41 browser details YourSeq 33 2582 2619 3000 86.2% chr2 - 127552178 127552213 36 browser details YourSeq 33 2579 2613 3000 97.2% chr2 - 39021142 39021176 35 browser details YourSeq 33 2580 2657 3000 69.6% chr14 - 18632616 18632691 76 browser details YourSeq 33 2572 2609 3000 94.8% chr11 - 61873976 61874017 42 browser details YourSeq 32 2573 2609 3000 94.6% chr8 - 87704666 87704706 41 browser details YourSeq 32 2578 2611 3000 97.1% chr5 - 135278434 135278467 34 browser details YourSeq 32 2573 2609 3000 94.6% chr15 - 37399415 37399454 40 browser details YourSeq 32 2573 2609 3000 94.6% chr11 - 30259836 30259876 41 browser details YourSeq 32 2578 2611 3000 97.1% chr1 - 133079288 133079321 34 browser details YourSeq 32 2574 2606 3000 100.0% chr3 + 85833818 85833853 36 browser details YourSeq 32 2573 2608 3000 97.3% chr2 + 23029926 23029967 42 browser details YourSeq 32 2579 2612 3000 97.1% chr16 + 58463296 58463329 34 browser details YourSeq 32 2573 2609 3000 97.2% chr15 + 79473161 79473203 43 browser details YourSeq 31 2579 2609 3000 100.0% chr2 + 132599816 132599846 31 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Pag1 phosphoprotein associated with glycosphingolipid microdomains 1 [ Mus musculus (house mouse) ] Gene ID: 94212, updated on 12-Aug-2019 Gene summary Official Symbol Pag1 provided by MGI Official Full Name phosphoprotein associated with glycosphingolipid microdomains 1 provided by MGI Primary source MGI:MGI:2443160 See related Ensembl:ENSMUSG00000027508 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 Cbp; Pag; F730007C19Rik Expression Broad expression in thymus adult (RPKM 20.1), spleen adult (RPKM 7.9) and 23 other tissues See more Orthologs human all Genomic context Location: 3; 3 A1 See Pag1 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (9687479..9833684, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (9691898..9833630, complement) Chromosome 3 - NC_000069.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Pag1 ENSMUSG00000027508 Description phosphoprotein associated with glycosphingolipid microdomains 1 [Source:MGI Symbol;Acc:MGI:2443160] Gene Synonyms Cbp, F730007C19Rik Location Chromosome 3: 9,687,479-9,833,679 reverse strand. GRCm38:CM000996.2 About this gene This gene has 5 transcripts (splice variants), 186 orthologues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pag1-204 ENSMUST00000161949.7 8256 429aa ENSMUSP00000124529.1 Protein coding CCDS17235 A6H659 Q3U1F9 TSL:1 GENCODE basic APPRIS P1 Pag1-201 ENSMUST00000108384.8 3729 429aa ENSMUSP00000104021.2 Protein coding CCDS17235 A6H659 Q3U1F9 TSL:1 GENCODE basic APPRIS P1 Pag1-203 ENSMUST00000159825.1 2406 No protein - Retained intron - - TSL:1 Pag1-205 ENSMUST00000163022.1 625 No protein - Retained intron - - TSL:2 Pag1-202 ENSMUST00000159497.1 1158 No protein - lncRNA - - TSL:1 166.20 kb Forward strand 9.70Mb 9.75Mb 9.80Mb Genes Gm16337-201 >lncRNA (Comprehensive set... Contigs < AC129945.10 AC156996.7 > Genes (Comprehensive set... < Pag1-204protein coding < Pag1-201protein coding < Pag1-203retained intron < Pag1-205retained intron < Gm24786-201rRNA < Pag1-202lncRNA Regulatory Build 9.70Mb 9.75Mb 9.80Mb Reverse strand 166.20 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000161949 < Pag1-204protein coding Reverse strand 146.20 kb ENSMUSP00000124... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 PANTHER PTHR16322:SF0 Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 429 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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