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Mouse Bcl3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Bcl3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Bcl3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Bcl3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Bcl3 gene (NCBI Reference Sequence: NM_033601 ; Ensembl: ENSMUSG00000053175 ) is located on Mouse chromosome 7. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000120537). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Bcl3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-33H23 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 lacking functional copies of this gene exhibit defects of the immune system including disruption of the humoral immune response and abnormal spleen and Peyer's patch organogenesis. Mutant mice show increased susceptibility to pathogens. Exon 3 starts from about 29.46% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 7533 bp, and the size of intron 3 for 3'-loxP site insertion: 705 bp. The size of effective cKO region: ~609 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 gRNA region 5' gRNA region 3' 1 3 4 5 6 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Bcl3 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(7109bp) | A(24.8% 1763) | C(25.94% 1844) | T(22.93% 1630) | G(26.33% 1872) 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 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% chr7 - 19812764 19815763 3000 browser details YourSeq 168 2728 2919 3000 92.7% chr7 - 19019044 19019234 191 browser details YourSeq 165 2728 2915 3000 92.5% chr5 + 135754375 135754560 186 browser details YourSeq 164 2728 2915 3000 92.6% chr7 - 17064288 17064474 187 browser details YourSeq 164 2728 2915 3000 92.5% chr5 - 136255247 136255432 186 browser details YourSeq 164 2728 2915 3000 93.5% chr7 + 24615899 24616085 187 browser details YourSeq 164 2728 2919 3000 91.7% chr7 + 16181103 16181293 191 browser details YourSeq 163 2727 2915 3000 92.1% chr7 - 19837327 19837514 188 browser details YourSeq 163 2728 2915 3000 93.7% chr5 - 140512278 140902159 389882 browser details YourSeq 163 2728 2915 3000 93.6% chr5 - 134043673 134375257 331585 browser details YourSeq 163 2728 2915 3000 93.6% chr7 + 28705846 28706137 292 browser details YourSeq 163 2728 2915 3000 93.6% chr7 + 18997259 19351507 354249 browser details YourSeq 162 2728 2915 3000 92.0% chr7 - 35765316 35765502 187 browser details YourSeq 162 2728 2915 3000 91.9% chr5 - 142808881 142809066 186 browser details YourSeq 162 2728 2915 3000 92.0% chr5 - 141782852 141783038 187 browser details YourSeq 162 2728 2915 3000 92.0% chr5 - 140484091 140484277 187 browser details YourSeq 162 2728 2915 3000 92.0% chr7 + 19757562 19757748 187 browser details YourSeq 162 2728 2915 3000 92.5% chr7 + 16264404 16264590 187 browser details YourSeq 162 2728 2919 3000 91.6% chr5 + 142849583 142849773 191 browser details YourSeq 162 2728 2915 3000 93.7% chr5 + 141088183 141088372 190 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% chr7 - 19809155 19812154 3000 browser details YourSeq 174 1321 1960 3000 85.6% chr11 - 97100558 97108089 7532 browser details YourSeq 134 1321 1610 3000 80.7% chr11 - 51910733 51911232 500 browser details YourSeq 133 1429 1770 3000 78.0% chr11 + 80245116 80245402 287 browser details YourSeq 109 1316 1785 3000 70.7% chr10 + 127389256 127389651 396 browser details YourSeq 108 1325 1802 3000 85.0% chr17 + 24024733 24025229 497 browser details YourSeq 104 1325 1808 3000 76.6% chr6 + 125199498 125199892 395 browser details YourSeq 104 1338 1809 3000 86.6% chr1 + 60534094 60534569 476 browser details YourSeq 100 1321 1776 3000 76.4% chr1 - 82818264 82818586 323 browser details YourSeq 92 1321 1802 3000 84.4% chr19 + 10626242 10679733 53492 browser details YourSeq 91 1665 1802 3000 83.9% chr11 - 32710573 32710710 138 browser details YourSeq 91 1535 1802 3000 86.4% chr1 - 14531877 14789743 257867 browser details YourSeq 91 1312 1806 3000 88.4% chr1 + 35940559 35941089 531 browser details YourSeq 90 1321 1739 3000 73.8% chr12 + 110416037 110416277 241 browser details YourSeq 85 1429 1613 3000 85.2% chr18 + 4123415 4123599 185 browser details YourSeq 77 1696 1802 3000 84.0% chr12 - 80965667 80965772 106 browser details YourSeq 77 1629 1797 3000 84.1% chrX + 162881828 162882201 374 browser details YourSeq 76 1339 1685 3000 74.8% chr7 + 105520887 105521008 122 browser details YourSeq 76 1696 1809 3000 82.2% chr18 + 36849820 36849932 113 browser details YourSeq 75 1321 1810 3000 82.9% chr11 + 70560021 70560506 486 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Bcl3 B cell leukemia/lymphoma 3 [ Mus musculus (house mouse) ] Gene ID: 12051, updated on 10-Oct-2019 Gene summary Official Symbol Bcl3 provided by MGI Official Full Name B cell leukemia/lymphoma 3 provided by MGI Primary source MGI:MGI:88140 See related Ensembl:ENSMUSG00000053175 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 Bcl-3; AI528691 Expression Biased expression in duodenum adult (RPKM 114.5), small intestine adult (RPKM 82.2) and 11 other tissues See more Orthologs human all Genomic context Location: 7 A3; 7 9.95 cM See Bcl3 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (19808462..19822824, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (20393811..20408104, complement) Chromosome 7 - NC_000073.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Bcl3 ENSMUSG00000053175 Description B cell leukemia/lymphoma 3 [Source:MGI Symbol;Acc:MGI:88140] Gene Synonyms Bcl-3 Location Chromosome 7: 19,808,462-19,822,770 reverse strand. GRCm38:CM001000.2 About this gene This gene has 6 transcripts (splice variants), 149 orthologues, 10 paralogues, is a member of 1 Ensembl protein family and is associated with 60 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Bcl3-201 ENSMUST00000120537.7 1850 448aa ENSMUSP00000113851.1 Protein coding CCDS20914 Q9Z2F6 TSL:1 GENCODE basic APPRIS P1 Bcl3-203 ENSMUST00000135609.7 755 114aa ENSMUSP00000117754.2 Protein coding - F6YNH8 CDS 5' incomplete TSL:3 Bcl3-202 ENSMUST00000123375.7 694 No protein - Retained intron - - TSL:3 Bcl3-206 ENSMUST00000152768.1 502 No protein - Retained intron - - TSL:3 Bcl3-204 ENSMUST00000139680.1 460 No protein - lncRNA - - TSL:3 Bcl3-205 ENSMUST00000141996.1 346 No protein - lncRNA - - TSL:3 34.31 kb Forward strand 19.80Mb 19.81Mb 19.82Mb 19.83Mb Genes Gm16175-201 >lncRNA Gm16174-201 >lncRNA (Comprehensive set... Contigs AC149282.2 > AC149085.6 > Genes (Comprehensive set... < Bcl3-201protein coding < Bcl3-203protein coding < Bcl3-206retained intron < Bcl3-202retained intron < Bcl3-205lncRNA < Bcl3-204lncRNA Regulatory Build 19.80Mb 19.81Mb 19.82Mb 19.83Mb Reverse strand 34.31 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000120537 < Bcl3-201protein coding Reverse strand 14.31 kb ENSMUSP00000113... MobiDB lite Low complexity (Seg) Superfamily Ankyrin repeat-containing domain superfamily SMART Ankyrin repeat Prints Ankyrin repeat Pfam Ankyrin repeat Ankyrin repeat-containing domain PROSITE profiles Ankyrin repeat-containing domain Ankyrin repeat PANTHER PTHR24118:SF51 PTHR24118 Gene3D Ankyrin repeat-containing domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 448 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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