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Mouse Osbpl8 Knockout Project (CRISPR/Cas9)

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Mouse Osbpl8 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Osbpl8 Knockout Project (CRISPR/Cas9) Objective: To create a Osbpl8 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Osbpl8 gene (NCBI Reference Sequence: NM_175489 ; Ensembl: ENSMUSG00000020189 ) is located on Mouse chromosome 10. 24 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 24 (Transcript: ENSMUST00000105275). Exon 3~4 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: Mice homozygous for a gene trap allele exhibit elevated of HDL and gender-specific alterations in lipid metabolism. Exon 3 starts from about 1.61% of the coding region. Exon 3~4 covers 6.56% of the coding region. The size of effective KO region: ~9019 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 3 4 24 Legends Exon of mouse Osbpl8 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 2000 bp section upstream of Exon 3 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 4 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(23.6% 472) | C(19.8% 396) | T(32.6% 652) | G(24.0% 480) Note: The 2000 bp section upstream of Exon 3 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(23.8% 476) | C(21.05% 421) | T(35.0% 700) | G(20.15% 403) Note: The 2000 bp section downstream of Exon 4 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 2000 1 2000 2000 100.0% chr10 + 111222673 111224672 2000 browser details YourSeq 62 528 708 2000 92.0% chr17 + 87413581 87414041 461 browser details YourSeq 54 577 688 2000 96.7% chr16 - 43967014 43967195 182 browser details YourSeq 49 570 703 2000 72.8% chr15 + 94467144 94467266 123 browser details YourSeq 47 617 703 2000 87.4% chr2 + 163942345 163942441 97 browser details YourSeq 44 621 704 2000 84.4% chr2 - 104690806 104691060 255 browser details YourSeq 44 601 679 2000 96.0% chr2 - 71351026 71351113 88 browser details YourSeq 44 604 706 2000 82.4% chr2 - 52662559 52662910 352 browser details YourSeq 42 624 704 2000 83.9% chr1 + 180720911 180721001 91 browser details YourSeq 41 651 716 2000 82.6% chr10 - 93687347 93687414 68 browser details YourSeq 40 651 703 2000 93.5% chr11 + 3596358 3596411 54 browser details YourSeq 39 653 703 2000 97.6% chr6 + 133964395 133964445 51 browser details YourSeq 37 655 703 2000 93.1% chr17 - 25167469 25167517 49 browser details YourSeq 37 653 704 2000 91.2% chr16 - 44477266 44477317 52 browser details YourSeq 37 651 703 2000 85.0% chr10 + 42452200 42452252 53 browser details YourSeq 36 653 704 2000 84.7% chr13 + 49291660 49291711 52 browser details YourSeq 36 653 704 2000 84.7% chr13 + 28890540 28890591 52 browser details YourSeq 35 657 707 2000 84.4% chr14 - 118476831 118476881 51 browser details YourSeq 35 651 703 2000 83.1% chr1 - 133939155 133939207 53 browser details YourSeq 35 651 703 2000 83.1% chr11 + 115908290 115908342 53 Note: The 2000 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 2000 1 2000 2000 100.0% chr10 + 111233692 111235691 2000 browser details YourSeq 37 766 842 2000 91.2% chr5 - 23843714 23843797 84 browser details YourSeq 25 193 217 2000 100.0% chr7 - 58516673 58516697 25 browser details YourSeq 22 1133 1154 2000 100.0% chr3 + 125260256 125260277 22 Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Osbpl8 oxysterol binding protein-like 8 [ Mus musculus (house mouse) ] Gene ID: 237542, updated on 10-Oct-2019 Gene summary Official Symbol Osbpl8 provided by MGI Official Full Name oxysterol binding protein-like 8 provided by MGI Primary source MGI:MGI:2443807 See related Ensembl:ENSMUSG00000020189 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as ORP-8; AA536976; AA536995; C730029P18Rik; D330025H14Rik Summary This gene encodes a member of the oxysterol-binding protein (Osbp) family, a group of intracellular lipid receptors. Like Expression most members, the encoded protein contains an N-terminal pleckstrin homology domain and a highly conserved C-terminal Osbp-like sterol-binding domain. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008] Orthologs Ubiquitous expression in CNS E18 (RPKM 7.5), frontal lobe adult (RPKM 6.6) and 25 other tissues See more human all Genomic context Location: 10; 10 D1 See Osbpl8 in Genome Data Viewer Exon count: 26 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (111164742..111297249) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (110601858..110734303) Chromosome 10 - NC_000076.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Osbpl8 ENSMUSG00000020189 Description oxysterol binding protein-like 8 [Source:MGI Symbol;Acc:MGI:2443807] Gene Synonyms D330025H14Rik, ORP-8 Location Chromosome 10: 111,164,752-111,297,249 forward strand. GRCm38:CM001003.2 About this gene This gene has 5 transcripts (splice variants), 206 orthologues, 11 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Osbpl8-202 ENSMUST00000105275.8 7204 889aa ENSMUSP00000100911.1 Protein coding CCDS36056 B9EJ86 TSL:1 GENCODE basic APPRIS ALT1 Osbpl8-201 ENSMUST00000095310.2 3622 847aa ENSMUSP00000092948.1 Protein coding CCDS24166 A0A0R4J150 TSL:1 GENCODE basic APPRIS P3 Osbpl8-205 ENSMUST00000238838.1 2523 695aa ENSMUSP00000158904.1 Protein coding - - CDS 3' incomplete Osbpl8-204 ENSMUST00000220139.1 2460 No protein - Retained intron - - TSL:1 Osbpl8-203 ENSMUST00000217693.1 624 No protein - lncRNA - - TSL:3 152.50 kb Forward strand 111.20Mb 111.25Mb 111.30Mb Genes (Comprehensive set... Gm8873-201 >processed pseudogene Osbpl8-203 >lncRNA Bbs10-202 >retained intron Osbpl8-202 >protein coding Osbpl8-201 >protein coding Osbpl8-204 >retained intron Bbs10-201 >protein coding Gm17849-201 >processed pseudogene Osbpl8-205 >protein coding Contigs AC124399.4 > < AC121871.3 Regulatory Build 111.20Mb 111.25Mb 111.30Mb Reverse strand 152.50 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000105275 132.50 kb Forward strand Osbpl8-202 >protein coding ENSMUSP00000100... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 Oxysterol-binding protein superfamily SMART Pleckstrin homology domain Pfam Pleckstrin homology domain Oxysterol-binding protein PROSITE profiles Pleckstrin homology domain PROSITE patterns Oxysterol-binding protein, conserved site PANTHER Oxysterol-binding protein PTHR10972:SF98 Gene3D PH-like domain superfamily 2.40.160.120 1.20.120.1290 CDD cd13286 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 800 889 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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