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Mouse Osbpl8 Conditional Knockout Project (CRISPR/Cas9)

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https://www.alphaknockout.com Mouse Osbpl8 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Osbpl8 conditional 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 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Osbpl8 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-456P17 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 gene trap allele exhibit elevated of HDL and gender-specific alterations in lipid metabolism. Exon 5 starts from about 8.17% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 21997 bp, and the size of intron 5 for 3'-loxP site insertion: 7421 bp. The size of effective cKO region: ~571 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 5 24 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Osbpl8 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(7071bp) | A(28.52% 2017) | C(20.24% 1431) | T(31.31% 2214) | G(19.93% 1409) 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% chr10 + 111252439 111255438 3000 browser details YourSeq 239 2528 2913 3000 87.0% chr2 - 80389043 80389472 430 browser details YourSeq 237 2543 2991 3000 86.9% chr11 - 94613676 94614153 478 browser details YourSeq 237 2598 3000 3000 84.5% chr12 + 117473501 117473920 420 browser details YourSeq 227 2528 2924 3000 81.7% chr16 + 34428838 34429264 427 browser details YourSeq 215 2595 2957 3000 81.7% chrX - 103884868 103885233 366 browser details YourSeq 211 2595 2924 3000 84.4% chr12 + 28921023 28921352 330 browser details YourSeq 208 2540 2914 3000 84.2% chr6 - 112418339 112418729 391 browser details YourSeq 207 2284 2943 3000 79.0% chr11 - 98861260 98861745 486 browser details YourSeq 202 2595 2939 3000 84.2% chr14 - 26966678 26967026 349 browser details YourSeq 201 2570 2924 3000 81.8% chr1 - 163605622 163605963 342 browser details YourSeq 197 2529 2924 3000 85.6% chr2 - 161631598 161632030 433 browser details YourSeq 196 2536 2922 3000 85.8% chr4_GL456350_random - 96523 96919 397 browser details YourSeq 196 2536 2922 3000 85.8% chr4 - 42474873 42475269 397 browser details YourSeq 194 2529 2951 3000 87.3% chr9 - 60556912 60557363 452 browser details YourSeq 194 2541 2945 3000 86.2% chr18 + 32807573 32808054 482 browser details YourSeq 193 2543 2943 3000 80.0% chr13 - 29132655 29133049 395 browser details YourSeq 192 2528 2920 3000 82.7% chr9 + 85875485 85875897 413 browser details YourSeq 192 2536 2922 3000 84.5% chr4 + 42371231 42371627 397 browser details YourSeq 192 2529 2920 3000 87.7% chr11 + 52863615 52864050 436 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% chr10 + 111256010 111259009 3000 browser details YourSeq 253 118 570 3000 82.0% chr2 - 117933524 117933968 445 browser details YourSeq 253 111 608 3000 83.8% chr1 + 128747150 128747642 493 browser details YourSeq 240 111 597 3000 85.0% chr8 + 125625174 125625641 468 browser details YourSeq 228 110 570 3000 83.0% chrX + 70960117 70960563 447 browser details YourSeq 227 113 608 3000 86.4% chr4 - 100703174 100703651 478 browser details YourSeq 226 204 592 3000 84.6% chr13 + 37098934 37099311 378 browser details YourSeq 223 114 592 3000 87.2% chr5 - 143155502 143155980 479 browser details YourSeq 217 113 606 3000 84.1% chr8 - 87135063 87135512 450 browser details YourSeq 215 114 565 3000 85.0% chr9 - 120255837 120256275 439 browser details YourSeq 213 113 616 3000 90.6% chr18 + 45446537 45492851 46315 browser details YourSeq 213 111 605 3000 88.1% chr17 + 84542540 84543047 508 browser details YourSeq 212 111 605 3000 82.0% chr2 - 154180962 154181403 442 browser details YourSeq 212 110 566 3000 89.3% chr8 + 57419158 57419659 502 browser details YourSeq 212 113 592 3000 86.1% chr13 + 91293721 91294195 475 browser details YourSeq 211 108 607 3000 87.3% chr1 + 24608306 24608800 495 browser details YourSeq 207 111 610 3000 89.1% chr18 - 39951548 39952068 521 browser details YourSeq 205 193 605 3000 88.4% chr3 - 133421673 133422088 416 browser details YourSeq 203 111 575 3000 85.1% chr15 + 9785101 9785562 462 browser details YourSeq 201 114 605 3000 85.7% chr4 + 12224307 12224793 487 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: 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 5 of 7 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 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000105275 132.50 kb Forward strand Osbpl8-202 >protein coding
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  • 393LN V 393P 344SQ V 393P Probe Set Entrez Gene

    393LN V 393P 344SQ V 393P Probe Set Entrez Gene

    393LN v 393P 344SQ v 393P Entrez fold fold probe set Gene Gene Symbol Gene cluster Gene Title p-value change p-value change chemokine (C-C motif) ligand 21b /// chemokine (C-C motif) ligand 21a /// chemokine (C-C motif) ligand 21c 1419426_s_at 18829 /// Ccl21b /// Ccl2 1 - up 393 LN only (leucine) 0.0047 9.199837 0.45212 6.847887 nuclear factor of activated T-cells, cytoplasmic, calcineurin- 1447085_s_at 18018 Nfatc1 1 - up 393 LN only dependent 1 0.009048 12.065 0.13718 4.81 RIKEN cDNA 1453647_at 78668 9530059J11Rik1 - up 393 LN only 9530059J11 gene 0.002208 5.482897 0.27642 3.45171 transient receptor potential cation channel, subfamily 1457164_at 277328 Trpa1 1 - up 393 LN only A, member 1 0.000111 9.180344 0.01771 3.048114 regulating synaptic membrane 1422809_at 116838 Rims2 1 - up 393 LN only exocytosis 2 0.001891 8.560424 0.13159 2.980501 glial cell line derived neurotrophic factor family receptor alpha 1433716_x_at 14586 Gfra2 1 - up 393 LN only 2 0.006868 30.88736 0.01066 2.811211 1446936_at --- --- 1 - up 393 LN only --- 0.007695 6.373955 0.11733 2.480287 zinc finger protein 1438742_at 320683 Zfp629 1 - up 393 LN only 629 0.002644 5.231855 0.38124 2.377016 phospholipase A2, 1426019_at 18786 Plaa 1 - up 393 LN only activating protein 0.008657 6.2364 0.12336 2.262117 1445314_at 14009 Etv1 1 - up 393 LN only ets variant gene 1 0.007224 3.643646 0.36434 2.01989 ciliary rootlet coiled- 1427338_at 230872 Crocc 1 - up 393 LN only coil, rootletin 0.002482 7.783242 0.49977 1.794171 expressed sequence 1436585_at 99463 BB182297 1 - up 393
  • Supplementary Table 1 Double Treatment Vs Single Treatment

    Supplementary Table 1 Double Treatment Vs Single Treatment

    Supplementary table 1 Double treatment vs single treatment Probe ID Symbol Gene name P value Fold change TC0500007292.hg.1 NIM1K NIM1 serine/threonine protein kinase 1.05E-04 5.02 HTA2-neg-47424007_st NA NA 3.44E-03 4.11 HTA2-pos-3475282_st NA NA 3.30E-03 3.24 TC0X00007013.hg.1 MPC1L mitochondrial pyruvate carrier 1-like 5.22E-03 3.21 TC0200010447.hg.1 CASP8 caspase 8, apoptosis-related cysteine peptidase 3.54E-03 2.46 TC0400008390.hg.1 LRIT3 leucine-rich repeat, immunoglobulin-like and transmembrane domains 3 1.86E-03 2.41 TC1700011905.hg.1 DNAH17 dynein, axonemal, heavy chain 17 1.81E-04 2.40 TC0600012064.hg.1 GCM1 glial cells missing homolog 1 (Drosophila) 2.81E-03 2.39 TC0100015789.hg.1 POGZ Transcript Identified by AceView, Entrez Gene ID(s) 23126 3.64E-04 2.38 TC1300010039.hg.1 NEK5 NIMA-related kinase 5 3.39E-03 2.36 TC0900008222.hg.1 STX17 syntaxin 17 1.08E-03 2.29 TC1700012355.hg.1 KRBA2 KRAB-A domain containing 2 5.98E-03 2.28 HTA2-neg-47424044_st NA NA 5.94E-03 2.24 HTA2-neg-47424360_st NA NA 2.12E-03 2.22 TC0800010802.hg.1 C8orf89 chromosome 8 open reading frame 89 6.51E-04 2.20 TC1500010745.hg.1 POLR2M polymerase (RNA) II (DNA directed) polypeptide M 5.19E-03 2.20 TC1500007409.hg.1 GCNT3 glucosaminyl (N-acetyl) transferase 3, mucin type 6.48E-03 2.17 TC2200007132.hg.1 RFPL3 ret finger protein-like 3 5.91E-05 2.17 HTA2-neg-47424024_st NA NA 2.45E-03 2.16 TC0200010474.hg.1 KIAA2012 KIAA2012 5.20E-03 2.16 TC1100007216.hg.1 PRRG4 proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) 7.43E-03 2.15 TC0400012977.hg.1 SH3D19