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Mouse Jpt2 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Jpt2 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Jpt2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Jpt2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Jpt2 gene (NCBI Reference Sequence: NM_198937.2 ; Ensembl: ENSMUSG00000024165 ) is located on Mouse chromosome 17. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000024981). 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 Jpt2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-40E8 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 2 starts from about 7.89% 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: 4401 bp, and the size of intron 2 for 3'-loxP site insertion: 7237 bp. The size of effective cKO region: ~649 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 gRNA region 5' gRNA region 3' 1 2 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Jpt2 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(7149bp) | A(27.36% 1956) | C(20.84% 1490) | T(29.71% 2124) | G(22.09% 1579) 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% chr17 - 24956417 24959416 3000 browser details YourSeq 228 807 2060 3000 92.0% chr12 - 111216611 111704428 487818 browser details YourSeq 174 808 1059 3000 90.0% chr8 + 66670765 66670982 218 browser details YourSeq 142 811 1747 3000 91.3% chr11 - 69503131 69725276 222146 browser details YourSeq 136 812 1061 3000 82.6% chr12 + 56506724 56506891 168 browser details YourSeq 135 636 950 3000 93.6% chr14 + 47606520 47606959 440 browser details YourSeq 131 577 945 3000 91.3% chr15 + 89409062 89409661 600 browser details YourSeq 129 674 940 3000 93.9% chr17 + 74369746 74370227 482 browser details YourSeq 127 806 947 3000 96.4% chr9 - 61896367 61896510 144 browser details YourSeq 127 796 947 3000 92.8% chr18 - 47385266 47385420 155 browser details YourSeq 126 808 950 3000 94.5% chr12 - 102372845 102372988 144 browser details YourSeq 124 811 973 3000 92.5% chr8 + 80675998 80676170 173 browser details YourSeq 123 796 947 3000 92.5% chr14 - 99260415 99260582 168 browser details YourSeq 122 797 945 3000 92.4% chr14 - 6794714 6794865 152 browser details YourSeq 122 806 945 3000 95.6% chr11 - 3432985 3433126 142 browser details YourSeq 122 811 950 3000 94.3% chr15 + 79594669 79594809 141 browser details YourSeq 122 808 950 3000 91.5% chr13 + 62514427 62514568 142 browser details YourSeq 121 812 945 3000 95.6% chrX - 161303323 161303457 135 browser details YourSeq 121 808 943 3000 95.6% chr12 - 15978970 15979109 140 browser details YourSeq 121 798 945 3000 92.4% chr2 + 154590324 154590474 151 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% chr17 - 24952768 24955767 3000 browser details YourSeq 210 1079 1481 3000 94.9% chr4 - 140978593 141291143 312551 browser details YourSeq 193 1085 1491 3000 94.1% chr4 - 126072836 126073399 564 browser details YourSeq 179 1340 1568 3000 92.9% chr5 - 137679131 137766929 87799 browser details YourSeq 160 1086 1608 3000 85.0% chr15 + 101113803 101114268 466 browser details YourSeq 158 1097 1480 3000 88.7% chr11 + 86070144 86282053 211910 browser details YourSeq 152 1341 1515 3000 94.8% chr4 - 78553428 78553603 176 browser details YourSeq 148 1 1138 3000 92.1% chrX + 160384586 160777413 392828 browser details YourSeq 146 1334 1507 3000 94.0% chr7 - 44235068 44235287 220 browser details YourSeq 139 1329 1481 3000 95.5% chr1 - 85744388 85744540 153 browser details YourSeq 138 1338 1493 3000 96.7% chr11 + 106436238 106436599 362 browser details YourSeq 134 1338 1479 3000 97.2% chr6 - 65763225 65763366 142 browser details YourSeq 130 1374 1573 3000 91.7% chr4 - 138974746 138975245 500 browser details YourSeq 128 1340 1479 3000 95.8% chr3 - 54670025 54670164 140 browser details YourSeq 127 1340 1478 3000 94.3% chr4 - 154889182 154889319 138 browser details YourSeq 126 1331 1478 3000 93.3% chr16 - 97733998 97734149 152 browser details YourSeq 124 1340 1486 3000 90.4% chr2 - 31796083 31796227 145 browser details YourSeq 123 1341 1473 3000 96.3% chr16 - 52541871 52542003 133 browser details YourSeq 122 1353 1493 3000 91.3% chr2 + 11551501 11551638 138 browser details YourSeq 120 1338 1465 3000 96.9% chr1 - 58456153 58456280 128 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: Jpt2 Jupiter microtubule associated homolog 2 [ Mus musculus (house mouse) ] Gene ID: 52009, updated on 26-Jun-2020 Gene summary Official Symbol Jpt2 provided by MGI Official Full Name Jupiter microtubule associated homolog 2 provided by MGI Primary source MGI:MGI:1196260 See related Ensembl:ENSMUSG00000024165 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Hn1l; AL022629; C16orf34; D17Ertd441e; 2810430B18Rik Expression Broad expression in CNS E11.5 (RPKM 42.7), placenta adult (RPKM 42.0) and 22 other tissues See more Orthologs human all Genomic context Location: 17 A3.3; 17 12.53 cM See Jpt2 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (24942470..24960623, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (25079415..25097568, complement) Chromosome 17 - NC_000083.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Jpt2 ENSMUSG00000024165 Description Jupiter microtubule associated homolog 2 [Source:MGI Symbol;Acc:MGI:1196260] Gene Synonyms 2810430B18Rik, D17Ertd441e, Hn1l Location Chromosome 17: 24,937,419-24,960,689 reverse strand. GRCm38:CM001010.2 About this gene This gene has 5 transcripts (splice variants), 263 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Jpt2- ENSMUST00000024981.8 2672 190aa ENSMUSP00000024981.7 Protein coding CCDS28504 Q3TM10 TSL:1 201 Q6PGH2 GENCODE basic APPRIS P1 Jpt2- ENSMUST00000233067.1 1430 190aa ENSMUSP00000156651.1 Nonsense mediated - Q3TM10 - 203 decay Q6PGH2 Jpt2- ENSMUST00000232978.1 501 No - Processed transcript - - - 202 protein Jpt2- ENSMUST00000233481.1 445 No - Processed transcript - - - 205 protein Jpt2- ENSMUST00000233316.1 1705 No - Retained intron - - - 204 protein Page 6 of 8 https://www.alphaknockout.com 43.27 kb Forward strand 24.93Mb 24.94Mb 24.95Mb 24.96Mb 24.97Mb Contigs < AC166102.2 AC154229.1 > Genes < Mapk8ip3-216nonsense mediated decay< Jpt2-203nonsense mediated decay < Cramp1l-203protein coding (Comprehensive set... < Mapk8ip3-221protein coding < Jpt2-201protein coding < Cramp1l-201protein coding < Mapk8ip3-203protein coding < Jpt2-202processed transcript < Mapk8ip3-201protein coding < Jpt2-204retained intron < Mapk8ip3-208protein coding < Jpt2-205processed transcript < Mapk8ip3-204protein coding < Mapk8ip3-202protein coding < Mapk8ip3-207protein coding < Mapk8ip3-205protein coding < Mapk8ip3-206protein coding < Mapk8ip3-218retained intron Regulatory Build 24.93Mb 24.94Mb 24.95Mb 24.96Mb 24.97Mb Reverse strand 43.27 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000024981 < Jpt2-201protein coding Reverse strand 18.22 kb ENSMUSP00000024... MobiDB lite PANTHER Microtubule-associated protein Jupiter PTHR34930:SF5 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 190 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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