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Mouse Timm50 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Timm50 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Timm50 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Timm50 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Timm50 gene (NCBI Reference Sequence: NM_025616 ; Ensembl: ENSMUSG00000003438 ) is located on Mouse chromosome 7. 11 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000081946). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Timm50 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-89A16 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 2 starts from about 10.29% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 905 bp, and the size of intron 4 for 3'-loxP site insertion: 1666 bp. The size of effective cKO region: ~1441 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 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Timm50 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7941bp) | A(22.3% 1771) | C(25.64% 2036) | T(26.84% 2131) | G(25.22% 2003) 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 - 28311270 28314269 3000 browser details YourSeq 342 306 851 3000 90.3% chr7 + 30202311 30202729 419 browser details YourSeq 333 143 781 3000 91.2% chr15 + 79073177 79074124 948 browser details YourSeq 323 142 756 3000 86.9% chr8 + 110896314 110896813 500 browser details YourSeq 298 319 840 3000 90.6% chr12 + 55411102 55411495 394 browser details YourSeq 293 319 836 3000 94.0% chr3 + 36562195 36562733 539 browser details YourSeq 289 319 789 3000 91.6% chr4 + 120488817 120489165 349 browser details YourSeq 279 319 829 3000 90.7% chr4 + 132662807 132663227 421 browser details YourSeq 266 319 808 3000 88.1% chr9 + 53602744 53603096 353 browser details YourSeq 255 142 512 3000 91.2% chr15 - 102391387 102391817 431 browser details YourSeq 247 147 507 3000 93.4% chr11 - 74709870 74710516 647 browser details YourSeq 246 142 508 3000 92.5% chr4 + 150278984 150279457 474 browser details YourSeq 243 164 505 3000 93.0% chr2 + 4894469 4895136 668 browser details YourSeq 242 313 788 3000 96.6% chr5 + 5702549 5703116 568 browser details YourSeq 231 244 776 3000 95.0% chr1 - 87824709 87825498 790 browser details YourSeq 227 358 847 3000 86.3% chr4 + 136300128 136300441 314 browser details YourSeq 226 143 525 3000 95.6% chr9 + 44296746 44297264 519 browser details YourSeq 224 321 746 3000 90.6% chr4 - 62337583 62337920 338 browser details YourSeq 223 318 845 3000 90.4% chr4 - 101819882 101820293 412 browser details YourSeq 223 319 825 3000 94.8% chr7 + 29237255 29237909 655 Note: The 3000 bp section upstream of Exon 2 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 - 28306829 28309828 3000 browser details YourSeq 105 41 543 3000 77.4% chr7 + 81569324 81569493 170 browser details YourSeq 97 379 537 3000 85.2% chr6 + 52405516 52405663 148 browser details YourSeq 97 382 532 3000 89.6% chr4 + 123165400 123165562 163 browser details YourSeq 93 369 521 3000 86.7% chr14 - 55867536 55867692 157 browser details YourSeq 91 383 531 3000 91.0% chr5 + 19791880 19792032 153 browser details YourSeq 91 402 519 3000 88.9% chr11 + 117794587 117794705 119 browser details YourSeq 88 397 521 3000 90.9% chr3 + 107212136 107212262 127 browser details YourSeq 87 377 520 3000 81.0% chr2 - 120908877 120909014 138 browser details YourSeq 87 379 536 3000 85.8% chr12 - 83610001 83610152 152 browser details YourSeq 87 402 537 3000 92.4% chr7 + 80016612 80017007 396 browser details YourSeq 87 391 518 3000 89.2% chr18 + 67336819 67336947 129 browser details YourSeq 86 401 537 3000 89.5% chr10 - 40817715 40817850 136 browser details YourSeq 85 382 537 3000 83.2% chr6 + 24776683 24776827 145 browser details YourSeq 85 403 536 3000 90.6% chr10 + 108588845 108588981 137 browser details YourSeq 84 396 537 3000 88.3% chr2 - 59773735 59773878 144 browser details YourSeq 84 402 536 3000 83.9% chr13 - 73958818 73958952 135 browser details YourSeq 84 407 541 3000 88.3% chr10 - 70295949 70296081 133 browser details YourSeq 84 397 558 3000 87.5% chr11 + 4413683 4413878 196 browser details YourSeq 84 397 532 3000 89.4% chr10 + 39902290 39902424 135 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Timm50 translocase of inner mitochondrial membrane 50 [ Mus musculus (house mouse) ] Gene ID: 66525, updated on 12-Aug-2019 Gene summary Official Symbol Timm50 provided by MGI Official Full Name translocase of inner mitochondrial membrane 50 provided by MGI Primary source MGI:MGI:1913775 See related Ensembl:ENSMUSG00000003438 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as TIM50L; AU015082; 2810403L02Rik Expression Ubiquitous expression in large intestine adult (RPKM 30.4), ovary adult (RPKM 29.7) and 28 other tissues See more Orthologs human all Genomic context Location: 7; 7 A3 See Timm50 in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (28305516..28312046, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (29090845..29097065, complement) Chromosome 7 - NC_000073.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Timm50 ENSMUSG00000003438 Description translocase of inner mitochondrial membrane 50 [Source:MGI Symbol;Acc:MGI:1913775] Gene Synonyms 2810403L02Rik, TIM50L Location Chromosome 7: 28,305,516-28,312,072 reverse strand. GRCm38:CM001000.2 About this gene This gene has 6 transcripts (splice variants), 185 orthologues, 5 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 Timm50-201 ENSMUST00000081946.4 1515 353aa ENSMUSP00000080614.4 Protein coding CCDS21039 Q9D880 TSL:1 GENCODE basic APPRIS P1 Timm50-202 ENSMUST00000131849.7 891 No protein - Retained intron - - TSL:2 Timm50-203 ENSMUST00000134263.1 632 No protein - Retained intron - - TSL:3 Timm50-206 ENSMUST00000151990.1 449 No protein - Retained intron - - TSL:1 Timm50-204 ENSMUST00000135220.1 394 No protein - Retained intron - - TSL:3 Timm50-205 ENSMUST00000136427.1 334 No protein - lncRNA - - TSL:1 26.56 kb Forward strand 28.30Mb 28.31Mb 28.32Mb Genes Gm44709-201 >lncRNA (Comprehensive set... Contigs AC148986.5 > Genes (Comprehensive set... < Dll3-201protein coding < Timm50-201protein coding < Supt5-202nonsense mediated decay < Dll3-203retained intron < Timm50-206retained intron < Timm50-204retained intron < Supt5-201protein coding < Timm50-203retained intron < Timm50-205lncRNA < Supt5-203retained intron < Timm50-202retained intron < Supt5-208protein coding < Supt5-207lncRNA Regulatory Build 28.30Mb 28.31Mb 28.32Mb Reverse strand 26.56 kb Regulation Legend CTCF 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: ENSMUST00000081946 < Timm50-201protein coding Reverse strand 6.56 kb ENSMUSP00000080... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily HAD-like superfamily SMART FCP1 homology domain Pfam FCP1 homology domain PROSITE profiles FCP1 homology domain PANTHER PTHR12210 Mitochondrial import inner membrane translocase subunit Tim50 Gene3D HAD superfamily CDD cd07521 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 353 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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