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Mouse Jph3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Jph3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Jph3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Jph3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Jph3 gene (NCBI Reference Sequence: NM_020605 ; Ensembl: ENSMUSG00000025318 ) is located on Mouse chromosome 8. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000026357). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Jph3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-177B2 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: Homozygotes for a targeted null mutation exhibit impaired balance and motor coordination. Exon 2 starts from about 17.16% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 21962 bp, and the size of intron 2 for 3'-loxP site insertion: 28416 bp. The size of effective cKO region: ~1278 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 2 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Jph3 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(7778bp) | A(22.38% 1741) | C(25.92% 2016) | T(24.12% 1876) | G(27.58% 2145) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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% chr8 + 121749718 121752717 3000 browser details YourSeq 176 1876 2767 3000 89.6% chr14 + 20368441 20646854 278414 browser details YourSeq 142 2608 2783 3000 91.4% chr11 + 101015468 101015648 181 browser details YourSeq 136 2604 2768 3000 92.6% chr10 + 76837259 76837438 180 browser details YourSeq 135 2607 2767 3000 92.6% chr3 - 40911059 40911222 164 browser details YourSeq 135 2619 2782 3000 92.5% chr11 - 62382978 62383142 165 browser details YourSeq 135 2585 2759 3000 90.9% chr15 + 58041320 58041530 211 browser details YourSeq 134 2607 2768 3000 90.7% chr18 + 14364113 14364273 161 browser details YourSeq 133 2607 2764 3000 89.7% chr12 - 54706692 54706846 155 browser details YourSeq 131 2607 2767 3000 92.8% chr15 - 99960706 99960971 266 browser details YourSeq 131 2607 2759 3000 90.6% chr6 + 116838377 116838526 150 browser details YourSeq 131 2608 2767 3000 88.5% chr10 + 121363242 121363397 156 browser details YourSeq 131 2612 2764 3000 90.0% chr10 + 24872243 24872391 149 browser details YourSeq 130 2628 2783 3000 93.4% chr7 - 122655290 122655448 159 browser details YourSeq 130 2607 2767 3000 89.2% chr11 - 118904530 118904687 158 browser details YourSeq 130 2607 2759 3000 94.0% chr10 - 93798191 93798343 153 browser details YourSeq 130 2607 2763 3000 89.7% chr11 + 33324906 33325060 155 browser details YourSeq 129 2610 2768 3000 91.6% chr1 - 101941460 101941624 165 browser details YourSeq 129 2620 2764 3000 93.1% chr5 + 143918651 143918794 144 browser details YourSeq 128 2607 2763 3000 91.1% chr13 - 64217433 64217589 157 Note: The 3000 bp section upstream of Exon 2 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% chr8 + 121753996 121756995 3000 browser details YourSeq 143 572 738 3000 92.9% chr3 + 88572516 88572682 167 browser details YourSeq 139 572 738 3000 95.0% chr2 - 121785864 121831256 45393 browser details YourSeq 139 558 738 3000 87.5% chr12 - 72634243 72634413 171 browser details YourSeq 137 558 737 3000 91.1% chr17 - 27670762 27670947 186 browser details YourSeq 136 558 738 3000 88.0% chr3 - 58319724 58319899 176 browser details YourSeq 135 558 738 3000 90.5% chr3 - 79969810 79969992 183 browser details YourSeq 134 572 740 3000 91.4% chr9 - 63913305 63913476 172 browser details YourSeq 134 558 732 3000 88.4% chrX + 58072174 58072344 171 browser details YourSeq 134 572 743 3000 90.9% chr11 + 29695692 29695866 175 browser details YourSeq 132 572 739 3000 89.8% chr17 - 29424881 29425051 171 browser details YourSeq 132 558 739 3000 90.4% chr17 - 13146501 13146683 183 browser details YourSeq 132 572 738 3000 91.8% chr17 + 85102733 85102903 171 browser details YourSeq 131 557 735 3000 89.3% chr7 - 58095802 58095978 177 browser details YourSeq 131 558 738 3000 90.2% chr17 - 25975287 25975467 181 browser details YourSeq 131 579 739 3000 90.7% chr4 + 150465195 150465355 161 browser details YourSeq 131 558 739 3000 89.1% chr3 + 152139200 152139377 178 browser details YourSeq 131 572 741 3000 90.2% chr15 + 70161006 70161178 173 browser details YourSeq 131 558 738 3000 89.8% chr11 + 99204955 99205135 181 browser details YourSeq 130 558 738 3000 84.3% chr5 - 124415696 124415869 174 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Jph3 junctophilin 3 [ Mus musculus (house mouse) ] Gene ID: 57340, updated on 12-Aug-2019 Gene summary Official Symbol Jph3 provided by MGI Official Full Name junctophilin 3 provided by MGI Primary source MGI:MGI:1891497 See related Ensembl:ENSMUSG00000025318 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 Jp3; JP-3 Expression Biased expression in cortex adult (RPKM 46.6), cerebellum adult (RPKM 35.8) and 5 other tissuesS ee more Orthologs human all Genomic context Location: 8; 8 E1 See Jph3 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (121730559..121791083) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (124254463..124314721) Chromosome 8 - NC_000074.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Jph3 ENSMUSG00000025318 Description junctophilin 3 [Source:MGI Symbol;Acc:MGI:1891497] Gene Synonyms JP-3 Location Chromosome 8: 121,729,623-121,794,276 forward strand. GRCm38:CM001001.2 About this gene This gene has 4 transcripts (splice variants), 188 orthologues, 3 paralogues, 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 Jph3- ENSMUST00000026357.11 3637 744aa ENSMUSP00000026357.5 Protein coding CCDS22728 Q9ET77 TSL:1 201 GENCODE basic APPRIS P1 Jph3- ENSMUST00000167439.1 2852 744aa ENSMUSP00000126190.1 Nonsense mediated - Q9ET77 TSL:1 202 decay Jph3- ENSMUST00000172209.1 679 No - Retained intron - - TSL:2 204 protein Jph3- ENSMUST00000169735.1 2092 No - lncRNA - - TSL:5 203 protein Page 6 of 8 https://www.alphaknockout.com 84.65 kb Forward strand 121.72Mb 121.74Mb 121.76Mb 121.78Mb 121.80Mb Genes Gm20388-201 >protein coding (Comprehensive set... Jph3-203 >lncRNA Jph3-201 >protein coding Jph3-202 >nonsense mediated decay Jph3-204 >retained intron Contigs AC103359.6 > AC121975.3 > Genes < Klhdc4-208nonsense mediated decay (Comprehensive set... < Klhdc4-209retained intron < Klhdc4-206retained intron < Klhdc4-204retained intron < Klhdc4-201protein coding < Klhdc4-205protein coding < Klhdc4-210protein coding < Klhdc4-203lncRNA Regulatory Build 121.72Mb 121.74Mb 121.76Mb 121.78Mb 121.80Mb Reverse strand 84.65 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000026357 60.26 kb Forward strand Jph3-201 >protein coding ENSMUSP00000026... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF82185 SMART MORN motif Pfam MORN motif PIRSF Junctophilin PANTHER Junctophilin PTHR23085:SF7 Gene3D 2.20.110.10 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 744 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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