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Mouse Tmem184b Knockout Project (CRISPR/Cas9)

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Mouse Tmem184b Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Tmem184b Knockout Project (CRISPR/Cas9) Objective: To create a Tmem184b knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tmem184b gene (NCBI Reference Sequence: NM_172608 ; Ensembl: ENSMUSG00000009035 ) is located on Mouse chromosome 15. 9 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 9 (Transcript: ENSMUST00000074991). Exon 4~6 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a gene-trapped allele exhibit delayed axon degeneration following peripheral nerve injury, progressive structural abnormalities at neuromuscular synapses, swellings within sensory terminals, sensory-motor dysfunction, and abnormal autophagy. Exon 4 starts from about 29.4% of the coding region. Exon 4~6 covers 21.21% of the coding region. The size of effective KO region: ~3402 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 4 5 6 9 Legends Exon of mouse Tmem184b Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 4 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 630 bp section downstream of Exon 6 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(21.65% 433) | C(25.7% 514) | T(26.35% 527) | G(26.3% 526) Note: The 2000 bp section upstream of Exon 4 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(630bp) | A(20.63% 130) | C(30.0% 189) | T(22.06% 139) | G(27.3% 172) Note: The 630 bp section downstream of Exon 6 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 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr15 - 79369989 79371988 2000 browser details YourSeq 182 597 946 2000 86.8% chr6 + 31594775 31595126 352 browser details YourSeq 176 597 947 2000 86.7% chr11 + 89203368 89203735 368 browser details YourSeq 168 599 947 2000 87.9% chr4 - 53724031 53724400 370 browser details YourSeq 168 599 938 2000 85.0% chr5 + 43342436 43342803 368 browser details YourSeq 168 596 947 2000 86.9% chr1 + 135291792 135292173 382 browser details YourSeq 163 596 947 2000 89.8% chr5 + 136898122 136898489 368 browser details YourSeq 161 648 945 2000 77.6% chr17 - 64128061 64128372 312 browser details YourSeq 159 597 947 2000 73.6% chr18 + 11551326 11551692 367 browser details YourSeq 155 661 943 2000 79.7% chr5 + 89297500 89407767 110268 browser details YourSeq 154 597 947 2000 82.4% chr16 + 22015326 22015660 335 browser details YourSeq 153 584 946 2000 79.8% chr11 - 43753327 43753637 311 browser details YourSeq 149 629 947 2000 87.9% chr2 - 84500145 84500467 323 browser details YourSeq 148 647 947 2000 88.2% chr8 - 105725946 105726259 314 browser details YourSeq 147 652 947 2000 85.8% chr8 - 126099650 126099954 305 browser details YourSeq 147 638 947 2000 77.8% chr5 - 116902113 116902383 271 browser details YourSeq 147 663 947 2000 83.7% chr8 + 124587801 124588083 283 browser details YourSeq 145 615 934 2000 86.5% chr11 + 114579739 114580084 346 browser details YourSeq 143 649 946 2000 90.1% chr5 + 150352398 150352696 299 browser details YourSeq 142 596 899 2000 87.4% chr17 - 15973352 15973777 426 Note: The 2000 bp section upstream of Exon 4 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 630 1 630 630 100.0% chr15 - 79365957 79366586 630 browser details YourSeq 27 469 506 630 93.6% chr13 - 74875237 74875275 39 browser details YourSeq 27 474 500 630 100.0% chr5 + 134604554 134604580 27 browser details YourSeq 21 1 23 630 95.7% chr1 - 157024571 157024593 23 browser details YourSeq 20 533 552 630 100.0% chr10 - 112209385 112209404 20 browser details YourSeq 20 487 506 630 100.0% chr10 + 66246811 66246830 20 Note: The 630 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Tmem184b transmembrane protein 184b [ Mus musculus (house mouse) ] Gene ID: 223693, updated on 24-Oct-2019 Gene summary Official Symbol Tmem184b provided by MGI Official Full Name transmembrane protein 184b provided by MGI Primary source MGI:MGI:2445179 See related Ensembl:ENSMUSG00000009035 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 2610507A11; 4732495E13Rik Expression Ubiquitous expression in lung adult (RPKM 33.0), adrenal adult (RPKM 25.5) and 26 other tissues See more Orthologs human all Genomic context Location: 15; 15 E1 See Tmem184b in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (79360684..79403303, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (79191114..79233733, complement) Chromosome 15 - NC_000081.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Tmem184b ENSMUSG00000009035 Description transmembrane protein 184b [Source:MGI Symbol;Acc:MGI:2445179] Gene Synonyms 4732495E13Rik Location Chromosome 15: 79,360,684-79,403,569 reverse strand. GRCm38:CM001008.2 About this gene This gene has 8 transcripts (splice variants), 245 orthologues, 4 paralogues, is a member of 2 Ensembl protein families and is associated with 18 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tmem184b- ENSMUST00000074991.9 3627 407aa ENSMUSP00000074518.2 Protein coding CCDS27639 Q8BG09 TSL:1 201 GENCODE basic APPRIS P2 Tmem184b- ENSMUST00000178522.2 3314 407aa ENSMUSP00000136416.1 Protein coding CCDS27639 Q8BG09 TSL:1 202 GENCODE basic APPRIS P2 Tmem184b- ENSMUST00000228002.1 3315 414aa ENSMUSP00000154210.1 Protein coding - A0A2I3BQI5 GENCODE 204 basic APPRIS ALT1 Tmem184b- ENSMUST00000231076.1 784 222aa ENSMUSP00000155585.1 Protein coding - A0A2R8VI00 CDS 3' 208 incomplete Tmem184b- ENSMUST00000228472.1 601 120aa ENSMUSP00000154053.1 Protein coding - A0A2I3BQ63 CDS 3' 205 incomplete Tmem184b- ENSMUST00000229049.1 361 44aa ENSMUSP00000155214.1 Protein coding - A0A2R8W6P8 CDS 3' 207 incomplete Tmem184b- ENSMUST00000226929.1 753 No - Retained - - - 203 protein intron Tmem184b- ENSMUST00000229003.1 625 No - Retained - - - 206 protein intron Page 7 of 9 https://www.alphaknockout.com 62.89 kb Forward strand 79.36Mb 79.37Mb 79.38Mb 79.39Mb 79.40Mb 79.41Mb Genes Maff-203 >protein coding (Comprehensive set... Maff-201 >protein coding Maff-202 >protein coding Maff-204 >protein coding Contigs < AL591913.14 Genes (Comprehensive set... < Tmem184b-201protein coding < Tmem184b-202protein coding < Tmem184b-204protein coding < Tmem184b-208protein coding < Tmem184b-203retained in<t rTomnem184b-207protein coding < Tmem184b-205protein coding < Mir1943-201miRNA < Tmem184b-206retained intron Regulatory Build 79.36Mb 79.37Mb 79.38Mb 79.39Mb 79.40Mb 79.41Mb Reverse strand 62.89 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000074991 < Tmem184b-201protein coding Reverse strand 42.89 kb ENSMUSP00000074... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Organic solute transporter subunit alpha/Transmembrane protein 184 PANTHER Organic solute transporter subunit alpha/Transmembrane protein 184 PTHR23423:SF28 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 407 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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