https://www.alphaknockout.com Mouse Itm2c Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Itm2c conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Itm2c gene (NCBI Reference Sequence: NM_022417 ; Ensembl: ENSMUSG00000026223 ) is located on Mouse chromosome 1. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000027425). Exon 3~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Itm2c gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-131A4 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, fertile and overtly normal with no alterations in the development, maturation and differentiation of B-lymphocytes or production of antibodies by antibody secreting cells. Exon 3 starts from about 33.21% of the coding region. The knockout of Exon 3~5 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 2063 bp, and the size of intron 5 for 3'-loxP site insertion: 388 bp. The size of effective cKO region: ~2389 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 2 3 4 5 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Itm2c 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(8833bp) | A(22.74% 2009) | C(25.81% 2280) | T(25.57% 2259) | G(25.87% 2285) 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% chr1 + 85901965 85904964 3000 browser details YourSeq 101 618 774 3000 82.1% chr7 - 4166714 4166864 151 browser details YourSeq 94 617 750 3000 81.6% chr18 - 78089002 78089131 130 browser details YourSeq 92 624 760 3000 81.5% chr9 + 52931941 52932075 135 browser details YourSeq 90 623 740 3000 88.9% chr10 - 90952980 90953098 119 browser details YourSeq 87 625 742 3000 85.3% chr4 - 98926155 98926270 116 browser details YourSeq 87 619 739 3000 91.6% chr13 - 38301641 38301763 123 browser details YourSeq 87 621 742 3000 88.5% chr11 + 78944230 79066530 122301 browser details YourSeq 86 627 742 3000 87.1% chr7 - 113634337 113634452 116 browser details YourSeq 86 616 742 3000 85.9% chr14 - 22670844 22670970 127 browser details YourSeq 85 623 749 3000 81.9% chr11 + 94379428 94379547 120 browser details YourSeq 83 618 721 3000 90.4% chr8 + 40516835 40516944 110 browser details YourSeq 82 623 742 3000 83.4% chr11 - 82498283 82498400 118 browser details YourSeq 82 616 742 3000 87.7% chr9 + 117792822 117792947 126 browser details YourSeq 82 612 760 3000 84.2% chr3 + 58225042 58225186 145 browser details YourSeq 81 625 762 3000 85.1% chr6 - 38762328 38762463 136 browser details YourSeq 81 625 778 3000 89.6% chr5 + 75875163 75875640 478 browser details YourSeq 81 613 721 3000 88.1% chr13 + 65290374 65290489 116 browser details YourSeq 81 614 753 3000 87.9% chr12 + 26871185 26871325 141 browser details YourSeq 80 623 721 3000 92.7% chr9 + 69657304 69657409 106 Note: The 3000 bp section upstream of Exon 3 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% chr1 + 85907354 85910353 3000 browser details YourSeq 83 2864 2980 3000 85.3% chr13 - 100801946 100802058 113 browser details YourSeq 80 2875 2995 3000 84.3% chr17 + 28745505 28745623 119 browser details YourSeq 76 2880 3000 3000 82.5% chr3 - 155341150 155341268 119 browser details YourSeq 76 2862 2974 3000 84.1% chr11 + 79923203 79923316 114 browser details YourSeq 74 2863 2969 3000 92.0% chr8 + 69857209 69857487 279 browser details YourSeq 72 2864 2982 3000 89.3% chr15 - 102297833 102297954 122 browser details YourSeq 72 2875 2972 3000 85.2% chr10 - 51066397 51066492 96 browser details YourSeq 71 2875 2972 3000 88.3% chr6 + 97193732 97193827 96 browser details YourSeq 71 2864 2973 3000 86.6% chr11 + 87347350 87347457 108 browser details YourSeq 71 2864 2969 3000 85.0% chr10 + 86141095 86141198 104 browser details YourSeq 70 2880 2995 3000 82.0% chr7 - 118617628 118617741 114 browser details YourSeq 70 2894 2997 3000 90.7% chr6 - 119911740 119911934 195 browser details YourSeq 70 2880 2973 3000 85.6% chr11 - 75507001 75507092 92 browser details YourSeq 69 2880 2977 3000 88.8% chr14 - 26594624 26594721 98 browser details YourSeq 69 2864 2972 3000 87.1% chr1 - 156548992 156549098 107 browser details YourSeq 69 2875 2973 3000 84.9% chr16 + 8608170 8608268 99 browser details YourSeq 69 2864 2996 3000 86.4% chr1 + 64435365 64647307 211943 browser details YourSeq 68 2864 2972 3000 88.0% chr17 - 24684127 24684233 107 browser details YourSeq 68 2880 2972 3000 88.8% chr11 - 53399230 53399320 91 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: Itm2c integral membrane protein 2C [ Mus musculus (house mouse) ] Gene ID: 64294, updated on 12-Aug-2019 Gene summary Official Symbol Itm2c provided by MGI Official Full Name integral membrane protein 2C provided by MGI Primary source MGI:MGI:1927594 See related Ensembl:ENSMUSG00000026223 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 E25; BRI3; E25C; ITM3; Bricd2c; 3110038L02Rik Expression Ubiquitous expression in adrenal adult (RPKM 251.4), cortex adult (RPKM 160.0) and 26 other tissues See more Orthologs human all Genomic context Location: 1; 1 C5 See Itm2c in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (85894510..85908698) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (87791085..87805273) Chromosome 1 - NC_000067.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Itm2c ENSMUSG00000026223 Description integral membrane protein 2C [Source:MGI Symbol;Acc:MGI:1927594] Gene Synonyms 3110038L02Rik, BRI3, Bricd2c, ITM3 Location Chromosome 1: 85,894,281-85,908,675 forward strand. GRCm38:CM000994.2 About this gene This gene has 3 transcripts (splice variants), 247 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Itm2c-201 ENSMUST00000027425.15 2230 269aa ENSMUSP00000027425.9 Protein coding CCDS35641 Q91VK4 TSL:1 GENCODE basic APPRIS P1 Itm2c-203 ENSMUST00000185569.1 1465 232aa ENSMUSP00000140692.1 Protein coding - A0A087WRM2 TSL:5 GENCODE basic Itm2c-202 ENSMUST00000139837.1 768 No protein - Retained intron - - TSL:2 34.40 kb Forward strand 85.89Mb 85.90Mb 85.91Mb Genes (Comprehensive set... Itm2c-201 >protein coding Itm2c-203 >protein coding Itm2c-202 >retained intron Contigs < AC107707.10 Regulatory Build 85.89Mb 85.90Mb 85.91Mb Reverse strand 34.40 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000027425 14.39 kb Forward strand Itm2c-201 >protein coding ENSMUSP00000027... Transmembrane heli... Low complexity (Seg) SMART BRICHOS domain Pfam BRICHOS domain PROSITE profiles BRICHOS domain PANTHER Integral membrane protein 2 PTHR10962:SF5 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 269 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.