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Mouse Trnp1 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Trnp1 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Trnp1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Trnp1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Trnp1 gene (NCBI Reference Sequence: NM_001081156 ; Ensembl: ENSMUSG00000056596 ) is located on Mouse chromosome 4. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000125541). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Trnp1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-392E4 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 1 starts from about 100% of the coding region. The knockout of Exon 1 will result in frameshift of the gene. The size of effective cKO region: ~840 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 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm cKO region Exon of mouse Trnp1 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(6923bp) | A(21.51% 1489) | C(25.13% 1740) | T(25.16% 1742) | G(28.2% 1952) 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% chr4 - 133498551 133501550 3000 browser details YourSeq 147 512 916 3000 93.0% chr7 - 29034252 29034973 722 browser details YourSeq 128 515 829 3000 89.0% chr9 - 75338434 75595431 256998 browser details YourSeq 127 682 918 3000 93.2% chr3 + 151763496 151763815 320 browser details YourSeq 120 669 893 3000 93.3% chr17 - 73465688 73466366 679 browser details YourSeq 113 682 905 3000 91.2% chr14 - 117370954 117371228 275 browser details YourSeq 109 682 919 3000 88.6% chr11 - 108134811 108135045 235 browser details YourSeq 109 682 872 3000 86.2% chr2 + 128741490 128741673 184 browser details YourSeq 108 747 933 3000 90.6% chr1 - 71733593 71733931 339 browser details YourSeq 107 682 919 3000 92.3% chr1 - 40341014 40341357 344 browser details YourSeq 107 669 933 3000 81.4% chr1 + 159757820 159757972 153 browser details YourSeq 104 682 921 3000 94.2% chr5 + 116810116 116810495 380 browser details YourSeq 103 754 919 3000 89.7% chr7 - 29034169 29034508 340 browser details YourSeq 102 742 910 3000 96.6% chr10 - 21177260 21177491 232 browser details YourSeq 102 757 919 3000 84.7% chr1 - 24331348 24331492 145 browser details YourSeq 98 754 919 3000 93.0% chr6 - 126189372 126189861 490 browser details YourSeq 97 682 915 3000 80.6% chr3 - 150146575 150146694 120 browser details YourSeq 95 682 916 3000 93.7% chr16 + 93984082 93984477 396 browser details YourSeq 95 740 928 3000 95.5% chr1 + 193496371 193496568 198 browser details YourSeq 94 742 885 3000 93.7% chr1 - 36968320 36968650 331 Note: The 3000 bp section upstream of Exon 1 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% chr4 - 133494628 133497627 3000 browser details YourSeq 43 1003 1071 3000 82.7% chr14 + 116022593 116022658 66 browser details YourSeq 40 1914 1974 3000 97.7% chr5 + 134455776 134455980 205 browser details YourSeq 38 1006 1067 3000 93.2% chr1 - 161490345 161490414 70 browser details YourSeq 32 997 1031 3000 87.9% chr17 + 59961445 59961477 33 browser details YourSeq 30 1002 1031 3000 100.0% chr3 + 156916474 156916503 30 browser details YourSeq 30 1002 1031 3000 100.0% chr14 + 11954944 11954973 30 browser details YourSeq 30 2632 2676 3000 86.2% chr11 + 97103323 97103366 44 browser details YourSeq 28 2643 2675 3000 93.8% chr16 + 56896101 56896133 33 browser details YourSeq 28 1958 1985 3000 100.0% chr11 + 68623429 68623456 28 browser details YourSeq 27 1004 1031 3000 100.0% chr15 - 7354308 7354342 35 browser details YourSeq 26 2618 2653 3000 86.2% chr19 - 40707901 40707936 36 browser details YourSeq 26 999 1031 3000 80.7% chr3 + 97048659 97048689 31 browser details YourSeq 25 2641 2671 3000 90.4% chr2 - 27832066 27832096 31 browser details YourSeq 25 1008 1034 3000 88.5% chr17 + 26998016 26998041 26 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Trnp1 TMF1-regulated nuclear protein 1 [ Mus musculus (house mouse) ] Gene ID: 69539, updated on 12-Aug-2019 Gene summary Official Symbol Trnp1 provided by MGI Official Full Name TMF1-regulated nuclear protein 1 provided by MGI Primary source MGI:MGI:1916789 See related Ensembl:ENSMUSG00000056596 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 Trnp; 2300002D11Rik Expression Biased expression in cerebellum adult (RPKM 22.7), frontal lobe adult (RPKM 21.3) and 13 other tissues See more Orthologs human all Genomic context Location: 4; 4 D2.3 See Trnp1 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (133491097..133499645, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (133047015..133054465, complement) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Trnp1 ENSMUSG00000056596 Description TMF1-regulated nuclear protein 1 [Source:MGI Symbol;Acc:MGI:1916789] Gene Synonyms 2300002D11Rik Location Chromosome 4: 133,491,100-133,498,550 reverse strand. GRCm38:CM000997.2 About this gene This gene has 2 transcripts (splice variants), 108 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Trnp1-202 ENSMUST00000125541.1 1654 223aa ENSMUSP00000129613.1 Protein coding CCDS38904 Q80ZI1 TSL:1 GENCODE basic APPRIS P1 Trnp1-201 ENSMUST00000123169.1 687 No protein - lncRNA - - TSL:2 27.45 kb Forward strand 133.485Mb 133.490Mb 133.495Mb 133.500Mb 133.505Mb Genes Tent5b-201 >protein coding (Comprehensive set... Contigs AL627228.31 > Genes (Comprehensive set... < Gm13257-201lncRNA < Trnp1-202protein coding < Trnp1-201lncRNA Regulatory Build 133.485Mb 133.490Mb 133.495Mb 133.500Mb 133.505Mb Reverse strand 27.45 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000125541 < Trnp1-202protein coding Reverse strand 7.45 kb ENSMUSP00000129... MobiDB lite Low complexity (Seg) PANTHER TMF-regulated nuclear protein 1 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 200 223 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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