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Mouse Tmem150a Conditional Knockout Project (CRISPR/Cas9)

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Mouse Tmem150a Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Tmem150a Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Tmem150a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tmem150a gene (NCBI Reference Sequence: NM_144916 ; Ensembl: ENSMUSG00000055912 ) is located on Mouse chromosome 6. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000069695). 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 Tmem150a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-180D9 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 100% 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: 586 bp, and the size of intron 2 for 3'-loxP site insertion: 409 bp. The size of effective cKO region: ~519 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 3 4 5 6 8 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Tmem150a 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(6973bp) | A(22.59% 1575) | C(27.79% 1938) | T(24.31% 1695) | G(25.31% 1765) 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 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% chr6 + 72352999 72355998 3000 browser details YourSeq 60 814 981 3000 94.3% chr17 + 67871036 67871282 247 browser details YourSeq 49 797 846 3000 100.0% chr13 - 52314281 52314338 58 browser details YourSeq 44 715 990 3000 94.2% chr11 - 87043485 87043761 277 browser details YourSeq 41 808 861 3000 78.6% chr1 + 127696904 127696945 42 browser details YourSeq 40 717 763 3000 95.6% chr17 + 88966729 88966797 69 browser details YourSeq 36 645 744 3000 87.5% chr11 - 53522474 53522571 98 browser details YourSeq 35 710 748 3000 94.9% chr8 + 117861946 117861984 39 browser details YourSeq 33 710 744 3000 97.2% chr1 - 185430239 185430273 35 browser details YourSeq 33 717 752 3000 97.3% chr11 + 118322440 118322477 38 browser details YourSeq 32 964 1007 3000 88.9% chr11 - 50657950 50657992 43 browser details YourSeq 30 710 743 3000 94.2% chr17 + 46055038 46055071 34 browser details YourSeq 28 717 744 3000 100.0% chr3 - 152403479 152403506 28 browser details YourSeq 28 710 741 3000 93.8% chr2 - 170113820 170113851 32 browser details YourSeq 27 963 989 3000 100.0% chr2 - 170189980 170190006 27 browser details YourSeq 27 963 1004 3000 83.9% chr10 - 99309630 99309669 40 browser details YourSeq 27 718 744 3000 100.0% chr3 + 90473210 90473236 27 browser details YourSeq 27 718 744 3000 100.0% chr2 + 31526251 31526277 27 browser details YourSeq 27 717 743 3000 100.0% chr10 + 61565396 61565422 27 browser details YourSeq 26 965 990 3000 100.0% chr8 - 45608691 45608716 26 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% chr6 + 72356518 72359517 3000 browser details YourSeq 46 882 931 3000 98.0% chr11 + 53814253 53814716 464 browser details YourSeq 39 2414 2461 3000 95.5% chr18 + 76041576 76041653 78 browser details YourSeq 38 882 931 3000 83.7% chr3 - 90520826 90520874 49 browser details YourSeq 37 881 931 3000 86.3% chr3 + 19290119 19290169 51 browser details YourSeq 36 868 931 3000 95.0% chr7 + 123083057 123083130 74 browser details YourSeq 36 895 982 3000 81.3% chr1 + 119576491 119576576 86 browser details YourSeq 35 881 931 3000 84.4% chr6 + 87450051 87450101 51 browser details YourSeq 34 898 931 3000 100.0% chrX + 53365807 53365840 34 browser details YourSeq 31 881 920 3000 94.5% chr13 + 49293697 49293736 40 browser details YourSeq 30 881 931 3000 96.9% chr8 + 3210160 3210211 52 browser details YourSeq 30 881 925 3000 88.3% chr11 + 32271541 32271584 44 browser details YourSeq 30 881 931 3000 90.7% chr10 + 69148444 69148493 50 browser details YourSeq 29 900 930 3000 96.8% chr8 - 115128861 115128891 31 browser details YourSeq 29 901 931 3000 96.8% chr7 + 65468456 65468486 31 browser details YourSeq 25 430 461 3000 96.3% chr3 - 136533653 136533686 34 browser details YourSeq 25 905 931 3000 96.3% chr19 - 46389691 46389717 27 browser details YourSeq 24 904 931 3000 92.9% chr11 + 12689249 12689276 28 browser details YourSeq 23 903 933 3000 87.1% chr1 + 157176956 157176986 31 browser details YourSeq 20 900 931 3000 81.3% chr2 + 84713700 84713731 32 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: Tmem150a transmembrane protein 150A [ Mus musculus (house mouse) ] Gene ID: 232086, updated on 12-Aug-2019 Gene summary Official Symbol Tmem150a provided by MGI Official Full Name transmembrane protein 150A provided by MGI Primary source MGI:MGI:2385244 See related Ensembl:ENSMUSG00000055912 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 Tmem150; BC014685 Expression Broad expression in placenta adult (RPKM 123.6), liver adult (RPKM 91.9) and 23 other tissues See more Orthologs human all Genomic context Location: 6; 6 C1 See Tmem150a in Genome Data Viewer Exon count: 8 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (72355483..72359762) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (72305477..72309756) Chromosome 6 - NC_000072.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Tmem150a ENSMUSG00000055912 Description transmembrane protein 150A [Source:MGI Symbol;Acc:MGI:2385244] Gene Synonyms Tmem150 Location Chromosome 6: 72,355,447-72,359,762 forward strand. GRCm38:CM000999.2 View alleles of this gene on alternative sequences About this gene This gene has 5 transcripts (splice variants), 1 gene allele, 174 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tmem150a- ENSMUST00000069695.8 1546 271aa ENSMUSP00000063977.2 Protein CCDS20239 Q91WN2 TSL:1 201 coding GENCODE basic APPRIS P1 Tmem150a- ENSMUST00000132243.2 1478 168aa ENSMUSP00000138445.1 Protein - S4R204 TSL:5 202 coding GENCODE basic Tmem150a- ENSMUST00000206531.1 544 174aa ENSMUSP00000145673.1 Protein - A0A0U1RNR4 CDS 3' 204 coding incomplete TSL:5 Tmem150a- ENSMUST00000206064.1 540 120aa ENSMUSP00000146268.1 Protein - A0A0U1RQ67 CDS 3' 203 coding incomplete TSL:5 Tmem150a- ENSMUST00000206821.1 551 No - lncRNA - - TSL:5 205 protein Page 6 of 8 https://www.alphaknockout.com 24.32 kb Forward strand 72.350Mb 72.355Mb 72.360Mb 72.365Mb Genes (Comprehensive set... 0610030E20Rik-204 >retained intron Tmem150a-202 >protein coding 0610030E20Rik-203 >retained intron Tmem150a-201 >protein coding 0610030E20Rik-201 >protein coding Tmem150a-203 >protein coding 0610030E20Rik-202 >retained intron Tmem150a-205 >lncRNA Tmem150a-204 >protein coding Contigs AC116115.11 > Genes < Gm45051-201lncRNA < Rnf181-201protein coding < Vamp5-203protein coding (Comprehensive set... < Rnf181-202protein coding < Vamp5-202protein coding < Rnf181-214retained intron < Vamp5-201protein coding < Rnf181-204nonsense mediated decay < Rnf181-207protein coding < Rnf181-212protein coding < Rnf181-210nonsense mediated decay < Rnf181-206nonsense mediated decay < Rnf181-205nonsense mediated decay < Rnf181-203protein coding < Rnf181-211nonsense mediated decay < Rnf181-209retained intron < Rnf181-208retained intron < Rnf181-213retained intron Regulatory Build 72.350Mb 72.355Mb 72.360Mb 72.365Mb Reverse strand 24.32 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000069695 4.32 kb Forward strand Tmem150a-201 >protein coding ENSMUSP00000063... Transmembrane heli... Low complexity (Seg) Pfam Frag1/DRAM/Sfk1 PANTHER PTHR21324 Transmembrane protein 150A All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 271 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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