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Mouse Tmtc2 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Tmtc2 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Tmtc2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Tmtc2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tmtc2 gene (NCBI Reference Sequence: NM_177368 ; Ensembl: ENSMUSG00000036019 ) is located on Mouse chromosome 10. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000061506). 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 Tmtc2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-34F6 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 3.35% 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: 159879 bp, and the size of intron 2 for 3'-loxP site insertion: 42438 bp. The size of effective cKO region: ~1071 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 gRNA region 5' gRNA region 3' 1 2 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Tmtc2 Homology arm 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(7571bp) | A(27.9% 2112) | C(19.67% 1489) | T(30.08% 2277) | G(22.36% 1693) 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 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% chr10 - 105414038 105417037 3000 browser details YourSeq 136 160 376 3000 87.3% chr2 + 107239977 107240209 233 browser details YourSeq 128 174 376 3000 84.8% chr17 - 48839892 48840089 198 browser details YourSeq 124 158 376 3000 88.2% chr15 - 94047851 94048084 234 browser details YourSeq 123 175 376 3000 87.5% chrX + 142176405 142176603 199 browser details YourSeq 118 178 370 3000 84.7% chr6 - 54607214 54607402 189 browser details YourSeq 118 175 376 3000 86.9% chr16 + 50006703 50006904 202 browser details YourSeq 116 175 376 3000 88.9% chr9 + 88091239 88091438 200 browser details YourSeq 114 175 376 3000 82.2% chr14 - 123334215 123334415 201 browser details YourSeq 112 175 374 3000 88.3% chr2 + 77874427 77874628 202 browser details YourSeq 109 178 374 3000 90.1% chr12 + 27527382 27527583 202 browser details YourSeq 108 175 376 3000 79.4% chrX - 105415200 105415385 186 browser details YourSeq 107 175 376 3000 85.5% chr2 + 126534809 126535006 198 browser details YourSeq 106 175 370 3000 85.3% chr17 - 69448089 69448278 190 browser details YourSeq 104 178 332 3000 81.7% chr9 - 20258435 20258587 153 browser details YourSeq 104 180 331 3000 85.5% chr8 - 81207811 81207955 145 browser details YourSeq 98 179 333 3000 82.0% chr1 + 106677600 106677756 157 browser details YourSeq 96 178 333 3000 86.9% chr7 - 89300027 89300184 158 browser details YourSeq 96 160 332 3000 86.5% chr10 - 107924280 107924462 183 browser details YourSeq 96 178 374 3000 86.3% chr18 + 68005076 68005337 262 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% chr10 - 105409967 105412966 3000 browser details YourSeq 133 1516 1879 3000 86.3% chr4 - 46974567 46974943 377 browser details YourSeq 131 1514 1789 3000 82.9% chr4 - 119000820 119001148 329 browser details YourSeq 122 1514 1694 3000 85.8% chr3 - 32429457 32429637 181 browser details YourSeq 117 1518 1789 3000 80.5% chr3 + 145835053 145835313 261 browser details YourSeq 112 1517 1693 3000 86.0% chr3 - 86275609 86275786 178 browser details YourSeq 101 1535 1696 3000 87.4% chr15 + 41771642 41771803 162 browser details YourSeq 100 1533 1787 3000 89.2% chr10 + 43156207 43156487 281 browser details YourSeq 99 1502 1674 3000 86.1% chr4 - 42371363 42371536 174 browser details YourSeq 99 1535 1760 3000 86.2% chr2 - 134612592 134612816 225 browser details YourSeq 99 1519 1881 3000 91.0% chr5 + 50440135 50440542 408 browser details YourSeq 99 1502 1674 3000 86.1% chr4 + 42803343 42803516 174 browser details YourSeq 97 1502 1694 3000 87.1% chr9 + 70620738 70620931 194 browser details YourSeq 97 1524 1696 3000 86.4% chr2 + 159490111 159490282 172 browser details YourSeq 94 1535 1694 3000 86.2% chr6 + 95522734 95522894 161 browser details YourSeq 94 1518 1789 3000 86.2% chr5 + 54578256 54578554 299 browser details YourSeq 92 1534 1692 3000 84.9% chr5 + 49061029 49061188 160 browser details YourSeq 91 1518 1676 3000 79.8% chr9 - 84592834 84592994 161 browser details YourSeq 90 1514 1805 3000 87.7% chr10 + 83971045 83971410 366 browser details YourSeq 89 1502 1652 3000 88.6% chr4_JH584294_random + 151138 151289 152 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Tmtc2 transmembrane and tetratricopeptide repeat containing 2 [ Mus musculus (house mouse) ] Gene ID: 278279, updated on 13-Aug-2019 Gene summary Official Symbol Tmtc2 provided by MGI Official Full Name transmembrane and tetratricopeptide repeat containing 2 provided by MGI Primary source MGI:MGI:1914057 See related Ensembl:ENSMUSG00000036019 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 8430438D04Rik; D330034A10Rik Expression Broad expression in limb E14.5 (RPKM 7.3), lung adult (RPKM 5.4) and 23 other tissues See more Orthologs human all Genomic context Location: 10; 10 D1 See Tmtc2 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (105187663..105574479, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (104624719..105011535, complement) Chromosome 10 - NC_000076.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Tmtc2 ENSMUSG00000036019 Description transmembrane and tetratricopeptide repeat containing 2 [Source:MGI Symbol;Acc:MGI:1914057] Gene Synonyms 8430438D04Rik, D330034A10Rik Location Chromosome 10: 105,187,663-105,574,451 reverse strand. GRCm38:CM001003.2 About this gene This gene has 6 transcripts (splice variants), 266 orthologues, 14 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tmtc2-201 ENSMUST00000061506.8 5629 836aa ENSMUSP00000061919.8 Protein coding CCDS24157 Q56A06 TSL:1 GENCODE basic APPRIS P1 Tmtc2-205 ENSMUST00000143691.1 4466 No protein - Retained intron - - TSL:1 Tmtc2-202 ENSMUST00000130665.1 2779 No protein - lncRNA - - TSL:1 Tmtc2-206 ENSMUST00000147997.1 924 No protein - lncRNA - - TSL:1 Tmtc2-203 ENSMUST00000133441.1 689 No protein - lncRNA - - TSL:3 Tmtc2-204 ENSMUST00000139741.1 624 No protein - lncRNA - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 406.79 kb Forward strand 105.2Mb 105.3Mb 105.4Mb 105.5Mb Genes Gm48206-201 >TEC Gm15664-201 >unprocessed pseudogene (Comprehensive set... Gm15663-202 >lncRNA Gm15663-201 >lncRNA Contigs AC132374.4 > AC121844.2 > < AC140299.2 Genes (Comprehensive set... < Tmtc2-201protein coding < Tmtc2-202lncRNA < Tmtc2-205retained intron < Tmtc2-206lncRNA < Gm48205-201TEC < Gm15666-201transcribed processed pseudogene < Tmtc2-203lncRNA < Gm48203-201TEC < Gm15666-202lncRNA < Tmtc2-204lncRNA < Gm48204-201TEC < Gm48207-201TEC Regulatory Build 105.2Mb 105.3Mb 105.4Mb 105.5Mb Reverse strand 406.79 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding pseudogene processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000061506 < Tmtc2-201protein coding Reverse strand 386.77 kb ENSMUSP00000061... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily Tetratricopeptide-like helical domain superfamily SMART Tetratricopeptide repeat Pfam Domain of unknown function DUF1736 PF13432 PF13424 Tetratricopeptide repeat 1 Tetratricopeptide repeat PROSITE profiles Tetratricopeptide repeat-containing domain Tetratricopeptide repeat PANTHER PTHR44216:SF3 PTHR44216 Gene3D Tetratricopeptide-like helical domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 836 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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