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Mouse Itih2 Knockout Project (CRISPR/Cas9)

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Mouse Itih2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Itih2 Knockout Project (CRISPR/Cas9) Objective: To create a Itih2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Itih2 gene (NCBI Reference Sequence: NM_010582 ; Ensembl: ENSMUSG00000037254 ) is located on Mouse chromosome 2. 21 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 21 (Transcript: ENSMUST00000042290). Exon 2~5 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 2 starts from about 3.4% of the coding region. Exon 2~5 covers 13.44% of the coding region. The size of effective KO region: ~5894 bp. The KO 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 3 4 5 21 Legends Exon of mouse Itih2 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1178 bp section upstream of Exon 2 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 5 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(1178bp) | A(34.38% 405) | C(19.27% 227) | T(29.2% 344) | G(17.15% 202) Note: The 1178 bp section upstream of Exon 2 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(31.4% 628) | C(18.9% 378) | T(24.05% 481) | G(25.65% 513) Note: The 2000 bp section downstream of Exon 5 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 1178 1 1178 1178 100.0% chr2 - 10129304 10130481 1178 browser details YourSeq 30 827 864 1178 94.2% chr11 - 86785721 86785764 44 browser details YourSeq 30 1035 1077 1178 94.5% chr10 - 9335180 9335226 47 browser details YourSeq 22 1034 1056 1178 100.0% chr1 - 176783874 176783902 29 browser details YourSeq 22 1091 1115 1178 95.9% chr1 - 41394317 41394342 26 Note: The 1178 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 2000 1 2000 2000 100.0% chr2 - 10121410 10123409 2000 browser details YourSeq 202 841 1139 2000 92.6% chr16 - 91111515 91111888 374 browser details YourSeq 177 841 1139 2000 88.3% chr1 + 130774041 130774252 212 browser details YourSeq 147 857 1138 2000 86.6% chr9 + 98156521 98156712 192 browser details YourSeq 146 840 1139 2000 97.5% chr3 + 81970762 81971096 335 browser details YourSeq 141 855 1139 2000 90.7% chr2 - 10760920 10761297 378 browser details YourSeq 130 932 1139 2000 94.1% chr6 + 40638195 40638428 234 browser details YourSeq 128 841 1109 2000 84.0% chr7 + 125269036 125269237 202 browser details YourSeq 127 852 1138 2000 95.3% chr2 + 5103435 5103755 321 browser details YourSeq 115 958 1136 2000 84.9% chr8 - 126385310 126385470 161 browser details YourSeq 109 886 1070 2000 82.9% chrX - 6485248 6485416 169 browser details YourSeq 109 845 1139 2000 93.6% chr15 + 89413231 89460294 47064 browser details YourSeq 107 976 1131 2000 91.7% chr17 - 27715165 27715451 287 browser details YourSeq 107 841 1130 2000 82.4% chr3 + 27796419 27796557 139 browser details YourSeq 105 998 1127 2000 97.4% chr4 + 99644020 99644385 366 browser details YourSeq 102 903 1036 2000 95.8% chr10 - 111104654 111104847 194 browser details YourSeq 94 841 1140 2000 79.1% chr5 - 56648139 56648244 106 browser details YourSeq 92 441 978 2000 82.1% chr2 + 164033314 164033910 597 browser details YourSeq 87 850 1069 2000 80.0% chr13 - 53756706 53756821 116 browser details YourSeq 86 423 575 2000 80.2% chr17 + 50804581 50804736 156 Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Itih2 inter-alpha trypsin inhibitor, heavy chain 2 [ Mus musculus (house mouse) ] Gene ID: 16425, updated on 12-Aug-2019 Gene summary Official Symbol Itih2 provided by MGI Official Full Name inter-alpha trypsin inhibitor, heavy chain 2 provided by MGI Primary source MGI:MGI:96619 See related Ensembl:ENSMUSG00000037254 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 Intin2; Itih-2; AI747202 Expression Biased expression in liver E18 (RPKM 372.8), liver E14 (RPKM 211.7) and 3 other tissues See more Orthologs human all Genomic context Location: 2 A1; 2 6.89 cM See Itih2 in Genome Data Viewer Exon count: 21 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (10094591..10130683, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (10016218..10052310, complement) Chromosome 2 - NC_000068.7 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Itih2 ENSMUSG00000037254 Description inter-alpha trypsin inhibitor, heavy chain 2 [Source:MGI Symbol;Acc:MGI:96619] Gene Synonyms Intin2, Itih-2 Location Chromosome 2: 10,094,593-10,131,396 reverse strand. GRCm38:CM000995.2 About this gene This gene has 3 transcripts (splice variants), 206 orthologues, 11 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Itih2-201 ENSMUST00000042290.13 3100 950aa ENSMUSP00000046530.7 Protein coding CCDS15677 G3X977 TSL:1 GENCODE basic APPRIS P1 Itih2-202 ENSMUST00000155809.8 1507 452aa ENSMUSP00000124636.1 Protein coding - Q3UEG7 CDS 3' incomplete TSL:1 Itih2-203 ENSMUST00000161909.1 391 81aa ENSMUSP00000124880.1 Protein coding - E0CX69 CDS 3' incomplete TSL:3 56.80 kb Forward strand 10.09Mb 10.10Mb 10.11Mb 10.12Mb 10.13Mb 10.14Mb Genes Kin-202 >lncRNA (Comprehensive set... Kin-201 >protein coding Kin-203 >lncRNA Contigs < AL772367.7 Genes (Comprehensive set... < Itih2-201protein coding < Itih2-202protein coding < Itih2-203protein coding Regulatory Build 10.09Mb 10.10Mb 10.11Mb 10.12Mb 10.13Mb 10.14Mb Reverse strand 56.80 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000042290 < Itih2-201protein coding Reverse strand 36.07 kb ENSMUSP00000046... Low complexity (Seg) Cleavage site (Sign... Superfamily von Willebrand factor A-like domain superfamily SMART VIT domain von Willebrand factor, type A Pfam VIT domain von Willebrand factor, type A Inter-alpha-trypsin inhibitor heavy chain, C-terminal PROSITE profiles VIT domain von Willebrand factor, type A PANTHER PTHR10338:SF14 PTHR10338 Gene3D von Willebrand factor A-like domain superfamily CDD cd01461 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 800 950 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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