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Mouse Timmdc1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Timmdc1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Timmdc1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Timmdc1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Timmdc1 gene (NCBI Reference Sequence: NM_024273 ; Ensembl: ENSMUSG00000002846 ) is located on Mouse chromosome 16. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000002925). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Timmdc1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-206M14 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 lethality. Heterozygous mice show an increased mean percentage of CD4 cells in the peripheral blood compared with controls, but no other notable heterozygous phenotype was detected. Exon 2 starts from about 22.81% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 3808 bp, and the size of intron 2 for 3'-loxP site insertion: 7559 bp. The size of effective cKO region: ~666 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 2 7 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Timmdc1 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(7166bp) | A(26.26% 1882) | C(19.89% 1425) | T(33.84% 2425) | G(20.01% 1434) 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% chr16 - 38518783 38521782 3000 browser details YourSeq 149 799 1836 3000 93.6% chr1 + 116189400 116466611 277212 browser details YourSeq 141 791 995 3000 89.2% chr4 + 43462561 43462746 186 browser details YourSeq 130 793 948 3000 95.0% chrX - 21698533 21698687 155 browser details YourSeq 127 791 946 3000 91.3% chr18 + 35691662 35691816 155 browser details YourSeq 125 778 1231 3000 81.7% chr1 - 164914113 164914330 218 browser details YourSeq 125 789 936 3000 95.7% chr15 + 58680200 58680356 157 browser details YourSeq 124 797 944 3000 93.5% chr2 + 121970341 121970487 147 browser details YourSeq 123 791 936 3000 95.6% chr10 - 40846214 40846359 146 browser details YourSeq 123 791 945 3000 89.7% chr3 + 83952283 83952437 155 browser details YourSeq 122 797 1244 3000 79.6% chr8 + 27788386 27788541 156 browser details YourSeq 122 791 945 3000 92.5% chr1 + 171468529 171468683 155 browser details YourSeq 121 791 944 3000 89.8% chr3 - 58331254 58331406 153 browser details YourSeq 121 793 936 3000 95.6% chr19 - 14462846 14462989 144 browser details YourSeq 121 797 945 3000 92.1% chr17 - 81293396 81293543 148 browser details YourSeq 121 793 945 3000 93.6% chr11 + 29227969 29228121 153 browser details YourSeq 120 791 939 3000 94.3% chr15 - 58131790 58131944 155 browser details YourSeq 120 798 940 3000 95.5% chr4 + 126993202 126993816 615 browser details YourSeq 120 797 940 3000 94.8% chr18 + 38986568 38986713 146 browser details YourSeq 119 791 947 3000 88.4% chr9 - 59738018 59738172 155 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% chr16 - 38515117 38518116 3000 browser details YourSeq 261 822 2427 3000 92.6% chr4 - 106355255 106612227 256973 browser details YourSeq 213 820 2436 3000 93.5% chr11 - 68988877 69240265 251389 browser details YourSeq 171 2707 3000 3000 88.4% chrX + 121333946 121334167 222 browser details YourSeq 166 1989 2436 3000 91.5% chrX - 151636309 151636900 592 browser details YourSeq 156 1976 2435 3000 81.2% chr15 - 90111595 90111900 306 browser details YourSeq 154 2244 2448 3000 88.9% chr5 + 23747044 23747251 208 browser details YourSeq 153 2243 2436 3000 89.7% chr5 - 92420912 92421110 199 browser details YourSeq 153 2185 2436 3000 89.6% chr17 - 70769063 70769680 618 browser details YourSeq 152 2243 2453 3000 85.1% chrX + 77415570 77415762 193 browser details YourSeq 152 2253 2437 3000 91.4% chr14 + 66032414 66032599 186 browser details YourSeq 150 2245 2437 3000 87.9% chr7 + 6898824 6899014 191 browser details YourSeq 148 2253 2437 3000 90.3% chr9 + 40701499 40701683 185 browser details YourSeq 147 2253 2435 3000 90.2% chr16 + 30953095 30953277 183 browser details YourSeq 146 2244 2434 3000 88.5% chr1 - 136012598 136012788 191 browser details YourSeq 146 2254 2436 3000 90.7% chr9 + 21534037 21534612 576 browser details YourSeq 146 2245 2435 3000 87.0% chr6 + 30086201 30086388 188 browser details YourSeq 145 2244 2436 3000 88.1% chr16 - 4505308 4505503 196 browser details YourSeq 144 1987 2437 3000 78.3% chr10 - 79924501 79924694 194 browser details YourSeq 144 2244 2436 3000 86.4% chr14 + 100449515 100449703 189 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Timmdc1 translocase of inner mitochondrial membrane domain containing 1 [ Mus musculus (house mouse) ] Gene ID: 76916, updated on 10-Oct-2019 Gene summary Official Symbol Timmdc1 provided by MGI Official Full Name translocase of inner mitochondrial membrane domain containing 1 provided by MGI Primary source MGI:MGI:1922139 See related Ensembl:ENSMUSG00000002846 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 AV135763; 2810021C21Rik; 4930455C21Rik Expression Ubiquitous expression in adrenal adult (RPKM 20.5), duodenum adult (RPKM 8.8) and 28 other tissues See more Orthologs human all Genomic context Location: 16; 16 B4 See Timmdc1 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (38497843..38522778, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (38497925..38522747, complement) Chromosome 16 - NC_000082.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Timmdc1 ENSMUSG00000002846 Description translocase of inner mitochondrial membrane domain containing 1 [Source:MGI Symbol;Acc:MGI:1922139] Gene Synonyms 2810021C21Rik, 4930455C21Rik Location Chromosome 16: 38,498,347-38,522,663 reverse strand. GRCm38:CM001009.2 About this gene This gene has 2 transcripts (splice variants), 189 orthologues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Timmdc1-201 ENSMUST00000002925.5 1573 285aa ENSMUSP00000002925.5 Protein coding CCDS28169 Q8BUY5 TSL:1 GENCODE basic APPRIS P1 Timmdc1-202 ENSMUST00000147543.1 678 No protein - lncRNA - - TSL:1 44.32 kb Forward strand 38.49Mb 38.50Mb 38.51Mb 38.52Mb 38.53Mb Genes Cd80-202 >protein coding Gm15953-201 >processed pseudogene (Comprehensive set... Contigs < AC209577.2 Genes < Timmdc1-201protein coding < Poglut1-201protein coding (Comprehensive set... < Timmdc1-202lncRNA < Poglut1-205retained intron < Poglut1-204retained intron Regulatory Build 38.49Mb 38.50Mb 38.51Mb 38.52Mb 38.53Mb Reverse strand 44.32 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000002925 < Timmdc1-201protein coding Reverse strand 24.32 kb ENSMUSP00000002... Transmembrane heli... MobiDB lite Pfam PF02466 PANTHER PTHR13002 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 285 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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