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Mouse Abtb1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Abtb1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Abtb1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Abtb1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Abtb1 gene (NCBI Reference Sequence: NM_030251 ; Ensembl: ENSMUSG00000030083 ) is located on Mouse chromosome 6. 12 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 12 (Transcript: ENSMUST00000032169). Exon 4~10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Abtb1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-56H21 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 4 starts from about 12.27% of the coding region. The knockout of Exon 4~10 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 938 bp, and the size of intron 10 for 3'-loxP site insertion: 1392 bp. The size of effective cKO region: ~2154 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 2 3 4 5 6 7 8 9 10 11 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Abtb1 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(8654bp) | A(19.25% 1666) | C(27.19% 2353) | T(24.16% 2091) | G(29.4% 2544) 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 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% chr6 - 88840022 88843021 3000 browser details YourSeq 40 2533 2699 3000 61.3% chr2 + 166857100 166857155 56 browser details YourSeq 33 2592 2692 3000 92.4% chr2 + 102082097 102082221 125 browser details YourSeq 33 501 548 3000 90.3% chr1 + 178568849 178569060 212 browser details YourSeq 22 519 540 3000 100.0% chr4 - 138969911 138969932 22 browser details YourSeq 22 2672 2693 3000 100.0% chr3 - 95255535 95255556 22 browser details YourSeq 22 2669 2690 3000 100.0% chr11 - 115725760 115725781 22 browser details YourSeq 21 2672 2692 3000 100.0% chr9 + 73016781 73016801 21 browser details YourSeq 21 2672 2692 3000 100.0% chr12 + 16860511 16860531 21 Note: The 3000 bp section upstream of Exon 4 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% chr6 - 88834868 88837867 3000 browser details YourSeq 27 839 869 3000 93.6% chr16 - 34309057 34309087 31 Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Abtb1 ankyrin repeat and BTB (POZ) domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 80283, updated on 13-Aug-2019 Gene summary Official Symbol Abtb1 provided by MGI Official Full Name ankyrin repeat and BTB (POZ) domain containing 1 provided by MGI Primary source MGI:MGI:1933148 See related Ensembl:ENSMUSG00000030083 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 BPOZ; EF1ABP; AI847549; BC003234 Expression Ubiquitous expression in thymus adult (RPKM 35.2), ovary adult (RPKM 26.5) and 28 other tissues See more Orthologs human all Genomic context Location: 6; 6 D1 See Abtb1 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (88835914..88842375, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (88785908..88791929, complement) Chromosome 6 - NC_000072.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 12 transcripts Gene: Abtb1 ENSMUSG00000030083 Description ankyrin repeat and BTB (POZ) domain containing 1 [Source:MGI Symbol;Acc:MGI:1933148] Gene Synonyms BPOZ, EF1ABP Location Chromosome 6: 88,835,914-88,841,984 reverse strand. GRCm38:CM000999.2 About this gene This gene has 12 transcripts (splice variants), 186 orthologues, 25 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Abtb1- ENSMUST00000032169.7 1845 478aa ENSMUSP00000032169.5 Protein coding CCDS20339 A0A0R4J0A1 TSL:1 201 GENCODE basic APPRIS P1 Abtb1- ENSMUST00000203272.2 4556 40aa ENSMUSP00000144802.1 Nonsense mediated - A0A0N4SVK7 TSL:1 204 decay Abtb1- ENSMUST00000203137.2 1774 36aa ENSMUSP00000144757.1 Nonsense mediated - A0A0N4SUP1 TSL:1 203 decay Abtb1- ENSMUST00000204458.2 783 40aa ENSMUSP00000145144.1 Nonsense mediated - A0A0N4SVK7 TSL:5 209 decay Abtb1- ENSMUST00000203864.2 645 43aa ENSMUSP00000145252.1 Nonsense mediated - A0A0N4SVV0 TSL:5 207 decay Abtb1- ENSMUST00000205082.2 596 47aa ENSMUSP00000144915.1 Nonsense mediated - A0A0N4SV19 CDS 5' 212 decay incomplete TSL:3 Abtb1- ENSMUST00000204327.2 579 22aa ENSMUSP00000145110.1 Nonsense mediated - A0A0N4SVH8 CDS 5' 208 decay incomplete TSL:3 Abtb1- ENSMUST00000204932.1 465 40aa ENSMUSP00000144922.1 Nonsense mediated - A0A0N4SVK7 TSL:2 211 decay Abtb1- ENSMUST00000203120.2 438 16aa ENSMUSP00000145338.1 Nonsense mediated - A0A0N4SW21 CDS 5' 202 decay incomplete TSL:3 Abtb1- ENSMUST00000204560.2 793 No - Retained intron - - TSL:2 210 protein Abtb1- ENSMUST00000203460.1 715 No - Retained intron - - TSL:3 205 protein Abtb1- ENSMUST00000203514.2 522 No - Retained intron - - TSL:3 206 protein Page 6 of 8 https://www.alphaknockout.com 26.07 kb Forward strand 88.83Mb 88.84Mb 88.85Mb Genes Mgll-204 >protein coding Gm15612-201 >lncRNA (Comprehensive set... Mgll-202 >protein coding Mgll-207 >protein coding Mgll-201 >protein coding Contigs AC153923.4 > Genes (Comprehensive set... < Abtb1-201protein coding < Podxl2-207lncRNA < Podxl2-212retained intron < Abtb1-203nonsense mediated decay < Podxl2-202protein coding < Abtb1-204nonsense mediated decay < Podxl2-201protein coding < Abtb1-210retained intron < Podxl2-210protein coding < Abtb1-202nonsense mediated decay < Podxl2-203retained intron < Podxl2-209protein coding < Abtb1-208nonsense mediated decay < Podxl2-206lncRNA < Abtb1-212nonsense mediated decay < Podxl2-205protein coding < Abtb1-209nonsense mediated decay < Podxl2-208lncRNA < Abtb1-206retained intron < Abtb1-207nonsense mediated decay < Abtb1-205retained intron < Abtb1-211nonsense mediated decay Regulatory Build 88.83Mb 88.84Mb 88.85Mb Reverse strand 26.07 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000032169 < Abtb1-201protein coding Reverse strand 5.99 kb ENSMUSP00000032... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Ankyrin repeat-containing domain superfamily SKP1/BTB/POZ domain superfamily SMART Ankyrin repeat BTB/POZ domain Pfam Ankyrin repeat-containing domain BTB/POZ domain PROSITE profiles Ankyrin repeat BTB/POZ domain Ankyrin repeat-containing domain PANTHER PTHR46231 Gene3D Ankyrin repeat-containing domain superfamily 3.30.710.10 CDD cd18295 cd18497 cd18296 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend frameshift variant missense variant splice region variant synonymous variant stop retained variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 478 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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