https://www.alphaknockout.com Mouse Dcbld2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Dcbld2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Dcbld2 gene (NCBI Reference Sequence: NM_028523 ; Ensembl: ENSMUSG00000035107 ) is located on Mouse chromosome 16. 16 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000046663). 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 Dcbld2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-86O4 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 exhibit reduced postnatal angiogenesis and impaired recovery from femoral artery ligation with impaired blood flow and decreased capillary density. Exon 5 starts from about 26.66% 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: 3059 bp, and the size of intron 7 for 3'-loxP site insertion: 884 bp. The size of effective cKO region: ~1888 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 5 6 7 8 9 16 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Dcbld2 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(8388bp) | A(26.13% 2192) | C(19.87% 1667) | T(32.61% 2735) | G(21.39% 1794) 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% chr16 + 58445203 58448202 3000 browser details YourSeq 149 825 1025 3000 90.1% chr5 - 147769073 147769252 180 browser details YourSeq 147 825 991 3000 96.9% chr4 - 59291648 59291816 169 browser details YourSeq 142 825 991 3000 93.4% chr10 + 16053684 16053844 161 browser details YourSeq 139 825 991 3000 95.9% chr17 - 51968195 51968359 165 browser details YourSeq 138 825 991 3000 95.2% chr3 - 140743622 140743786 165 browser details YourSeq 137 845 991 3000 98.0% chr11 + 112734954 112735113 160 browser details YourSeq 135 825 991 3000 91.6% chr9 - 47995644 47995796 153 browser details YourSeq 135 845 991 3000 97.9% chr4 - 58322789 58323034 246 browser details YourSeq 134 825 991 3000 90.8% chr1 - 22327761 22327909 149 browser details YourSeq 133 845 991 3000 97.2% chr8 - 95515476 95515643 168 browser details YourSeq 133 845 991 3000 93.4% chr10 + 66495089 66495224 136 browser details YourSeq 132 845 991 3000 98.6% chr9 + 48217646 48217817 172 browser details YourSeq 128 825 991 3000 97.1% chr6 + 32110081 32110253 173 browser details YourSeq 126 825 991 3000 94.4% chrY + 77732162 77732378 217 browser details YourSeq 126 825 991 3000 94.4% chrY + 65416094 65416318 225 browser details YourSeq 126 825 991 3000 94.4% chrY + 54693978 54694194 217 browser details YourSeq 125 853 991 3000 97.8% chr8 - 77820822 77820975 154 browser details YourSeq 122 1827 1989 3000 90.1% chr7 - 58629345 58629508 164 browser details YourSeq 122 871 1003 3000 97.7% chr6 - 138050451 138050628 178 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% chr16 + 58450091 58453090 3000 browser details YourSeq 44 2483 2536 3000 95.9% chr6 + 99504441 99504498 58 browser details YourSeq 42 2486 2552 3000 74.0% chr15 + 36196369 36196417 49 browser details YourSeq 31 2790 2820 3000 100.0% chr18 + 86972892 86972922 31 browser details YourSeq 31 2473 2508 3000 97.2% chr10 + 113763670 113763719 50 Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Dcbld2 discoidin, CUB and LCCL domain containing 2 [ Mus musculus (house mouse) ] Gene ID: 73379, updated on 14-Aug-2019 Gene summary Official Symbol Dcbld2 provided by MGI Official Full Name discoidin, CUB and LCCL domain containing 2 provided by MGI Primary source MGI:MGI:1920629 See related Ensembl:ENSMUSG00000035107 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 Esdn; CLCP1; AW146002; 1700055P21Rik Expression Ubiquitous expression in bladder adult (RPKM 10.6), CNS E11.5 (RPKM 10.5) and 27 other tissues See more Orthologs human all Genomic context Location: 16; 16 C1.2 See Dcbld2 in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (58408535..58469745) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (58408648..58469858) Chromosome 16 - NC_000082.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Dcbld2 ENSMUSG00000035107 Description discoidin, CUB and LCCL domain containing 2 [Source:MGI Symbol;Acc:MGI:1920629] Gene Synonyms 1700055P21Rik, CLCP1, Esdn Location Chromosome 16: 58,408,443-58,469,727 forward strand. GRCm38:CM001009.2 About this gene This gene has 7 transcripts (splice variants), 208 orthologues, 35 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Dcbld2-201 ENSMUST00000046663.7 6561 769aa ENSMUSP00000039915.7 Protein coding CCDS28229 Q91ZV3 TSL:1 GENCODE basic APPRIS P1 Dcbld2-202 ENSMUST00000130409.7 2396 No protein - Retained intron - - TSL:1 Dcbld2-204 ENSMUST00000135415.1 2359 No protein - Retained intron - - TSL:1 Dcbld2-203 ENSMUST00000134324.7 891 No protein - Retained intron - - TSL:3 Dcbld2-207 ENSMUST00000150817.7 1270 No protein - lncRNA - - TSL:1 Dcbld2-205 ENSMUST00000142830.7 627 No protein - lncRNA - - TSL:3 Dcbld2-206 ENSMUST00000149321.1 576 No protein - lncRNA - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 81.28 kb Forward strand 58.40Mb 58.42Mb 58.44Mb 58.46Mb Genes (Comprehensive set... Dcbld2-201 >protein coding Dcbld2-205 >lncRNA Dcbld2-206 >lncRNA Dcbld2-203 >retained intron Dcbld2-207 >lncRNA Dcbld2-202 >retained intron Dcbld2-204 >retained intron Contigs CT027564.7 > Genes < 4930461C15Rik-201lncRNA < St3gal6-207protein coding (Comprehensive set... < St3gal6-202protein coding < St3gal6-201protein coding < St3gal6-212lncRNA Regulatory Build 58.40Mb 58.42Mb 58.44Mb 58.46Mb Reverse strand 81.28 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 processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000046663 61.28 kb Forward strand Dcbld2-201 >protein coding ENSMUSP00000039... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily Galactose-binding-like domain superfamily LCCL domain superfamily Spermadhesin, CUB domain superfamily SMART CUB domain LCCL domain Coagulation factor 5/8 C-terminal domain Pfam CUB domain LCCL domain Coagulation factor 5/8 C-terminal domain PROSITE profiles CUB domain LCCL domain Coagulation factor 5/8 C-terminal domain PROSITE patterns Coagulation factor 5/8 C-terminal domain PANTHER PTHR46806:SF3 PTHR46806 Gene3D Spermadhesin, CUB domain superfamily Galactose-binding-like domain superfamily LCCL domain superfamily CDD CUB domain Coagulation factor 5/8 C-terminal domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe insertion missense variant splice region variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 769 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.