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Mouse Ppie Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ppie Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ppie Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ppie conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ppie gene (NCBI Reference Sequence: NM_019489 ; Ensembl: ENSMUSG00000028651 ) is located on Mouse chromosome 4. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000030404). Exon 5~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppie gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-383B3 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: Exon 5 starts from about 22.37% of the coding region. The knockout of Exon 5~7 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1194 bp, and the size of intron 7 for 3'-loxP site insertion: 2679 bp. The size of effective cKO region: ~2944 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 5' gRNA region gRNA region 3' 1 3 4 5 6 7 10 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ppie 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(9444bp) | A(25.37% 2396) | C(21.66% 2046) | T(30.65% 2895) | G(22.31% 2107) 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% chr4 - 123135928 123138927 3000 browser details YourSeq 170 719 1154 3000 90.5% chr10 + 40341198 40341692 495 browser details YourSeq 161 714 1144 3000 84.6% chr4 + 124734091 124734277 187 browser details YourSeq 160 716 1144 3000 90.9% chr9 + 59646420 59646860 441 browser details YourSeq 153 716 1143 3000 84.6% chr2 + 167665867 167666090 224 browser details YourSeq 153 700 1144 3000 84.0% chr12 + 117609306 117609593 288 browser details YourSeq 152 967 1144 3000 94.2% chr2 - 167998817 167998994 178 browser details YourSeq 151 715 1144 3000 84.0% chr2 - 128640842 128641030 189 browser details YourSeq 151 963 1144 3000 92.0% chr11 - 59391692 59391872 181 browser details YourSeq 151 963 1144 3000 94.2% chr2 + 137636515 137637016 502 browser details YourSeq 150 969 1144 3000 92.9% chr10 - 8180226 8180400 175 browser details YourSeq 149 971 1149 3000 93.1% chr13 - 91440612 91440790 179 browser details YourSeq 148 979 1144 3000 96.3% chr7 - 75452021 75452186 166 browser details YourSeq 148 968 1144 3000 92.1% chr2 - 172227657 172228100 444 browser details YourSeq 148 714 1144 3000 93.1% chr18 - 67677332 67677911 580 browser details YourSeq 148 715 1144 3000 82.3% chr2 + 144702748 144702936 189 browser details YourSeq 147 994 1267 3000 93.5% chr11 - 77510900 77511210 311 browser details YourSeq 147 974 1144 3000 94.6% chr6 + 31652093 31652263 171 browser details YourSeq 147 973 1144 3000 94.6% chr11 + 117065599 117065771 173 browser details YourSeq 146 962 1144 3000 94.0% chr12 - 59113698 59113958 261 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% chr4 - 123129984 123132983 3000 browser details YourSeq 102 621 801 3000 89.4% chr4 + 141211968 141594827 382860 browser details YourSeq 83 624 776 3000 92.2% chr11 + 23070010 23070454 445 browser details YourSeq 76 623 767 3000 93.3% chr13 - 119816173 119816590 418 browser details YourSeq 75 606 874 3000 91.3% chr15 - 47498354 47498791 438 browser details YourSeq 75 617 772 3000 92.4% chr19 + 25377864 25378081 218 browser details YourSeq 74 512 697 3000 89.4% chr11 - 29557319 29557507 189 browser details YourSeq 72 617 1141 3000 72.1% chr17 - 6633566 6633838 273 browser details YourSeq 71 575 690 3000 89.9% chr19 - 7565145 7565481 337 browser details YourSeq 69 614 717 3000 88.0% chr16 + 36472971 36473076 106 browser details YourSeq 68 596 695 3000 93.6% chr11 - 97633013 97633523 511 browser details YourSeq 67 607 697 3000 92.5% chr2 - 41076161 41076269 109 browser details YourSeq 66 621 706 3000 91.4% chr5 + 126148030 126148118 89 browser details YourSeq 66 617 695 3000 92.4% chr4 + 88649639 88649718 80 browser details YourSeq 66 618 713 3000 85.6% chr12 + 84098545 84098641 97 browser details YourSeq 65 638 713 3000 93.4% chr7 + 27583265 27583341 77 browser details YourSeq 64 614 695 3000 87.2% chr5 + 134317977 134318053 77 browser details YourSeq 64 617 697 3000 95.8% chr17 + 68564923 68565004 82 browser details YourSeq 63 614 695 3000 94.4% chr3 - 35655759 35655841 83 browser details YourSeq 63 620 700 3000 95.7% chr2 - 24582461 24582542 82 Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ppie peptidylprolyl isomerase E (cyclophilin E) [ Mus musculus (house mouse) ] Gene ID: 56031, updated on 12-Aug-2019 Gene summary Official Symbol Ppie provided by MGI Official Full Name peptidylprolyl isomerase E (cyclophilin E) provided by MGI Primary source MGI:MGI:1917118 See related Ensembl:ENSMUSG00000028651 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 Cyp33; 2010010D16Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 21.0), limb E14.5 (RPKM 14.1) and 28 other tissues See more Orthologs human all Genomic context Location: 4; 4 D2.2 See Ppie in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (123127114..123139990, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (122804368..122817184, complement) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Ppie ENSMUSG00000028651 Description peptidylprolyl isomerase E (cyclophilin E) [Source:MGI Symbol;Acc:MGI:1917118] Gene Synonyms 2010010D16Rik Location Chromosome 4: 123,127,115-123,139,951 reverse strand. GRCm38:CM000997.2 About this gene This gene has 4 transcripts (splice variants), 185 orthologues, 17 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ppie-201 ENSMUST00000030404.4 1188 301aa ENSMUSP00000030404.4 Protein coding CCDS18610 Q9QZH3 TSL:1 GENCODE basic APPRIS P1 Ppie-203 ENSMUST00000136466.7 861 No protein - lncRNA - - TSL:5 Ppie-202 ENSMUST00000126558.7 804 No protein - lncRNA - - TSL:2 Ppie-204 ENSMUST00000137778.1 669 No protein - lncRNA - - TSL:5 32.84 kb Forward strand 123.12Mb 123.13Mb 123.14Mb Genes Oxct2b-201 >protein coding (Comprehensive set... Bmp8b-201 >protein coding Bmp8b-202 >lncRNA Contigs AL606934.12 > Genes (Comprehensive set... < Ppie-201protein coding < Ppie-203lncRNA < Ppie-202lncRNA < Ppie-204lncRNA Regulatory Build 123.12Mb 123.13Mb 123.14Mb Reverse strand 32.84 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000030404 < Ppie-201protein coding Reverse strand 12.84 kb protein_pic We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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