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Mouse Tmem209 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Tmem209 Conditional Knockout Project (CRISPR/Cas9) http://www.alphaknockout.com/ Mouse Tmem209 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Tmem209 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tmem209 gene ( NCBI Reference Sequence: NM_178625 ; Ensembl: ENSMUSG00000029782 ) is located on mouse chromosome 6. 15 exons are identified , with the ATG start codon in exon 1 and the TAG stop codon in exon 15 (Transcript Tmem209-204: ENSMUST00000115160). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the mouse Tmem209 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-317G5 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: The knockout of Exon 4~5 will result in frameshift of the gene, and covers 22.22% of the coding region. The size of effective cKO region: ~2246 bp. Page 1 of 8 http://www.alphaknockout.com/ Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 15 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Tmem209 Homology arm cKO region loxP site Page 2 of 8 http://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(8185bp) | A(28.42% 2326) | C(20.27% 1659) | G(21.61% 1769) | T(29.7% 2431) 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 http://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 - 30507168 30510167 3000 browser details YourSeq 148 2382 3000 3000 83.5% chr11 + 97271861 97272261 401 browser details YourSeq 128 2500 3000 3000 91.7% chr2 - 11608644 11609246 603 browser details YourSeq 128 2860 3000 3000 96.5% chr15 - 80951401 80951544 144 browser details YourSeq 127 2847 3000 3000 89.1% chr9 + 61059119 61059266 148 browser details YourSeq 122 2859 3000 3000 94.9% chr12 - 116787847 116787989 143 browser details YourSeq 122 2850 3000 3000 93.0% chr14 + 20455033 20455186 154 browser details YourSeq 120 2856 3000 3000 93.6% chr17 - 88151556 88151702 147 browser details YourSeq 120 2463 3000 3000 83.5% chr8 + 31069958 31070400 443 browser details YourSeq 120 2857 3000 3000 93.1% chr5 + 27813829 27813987 159 browser details YourSeq 120 2859 3000 3000 92.3% chr4 + 140687606 140687747 142 browser details YourSeq 120 2868 3000 3000 95.5% chr1 + 68964934 68965066 133 browser details YourSeq 119 2856 3000 3000 91.6% chr9 - 110138281 110138427 147 browser details YourSeq 119 2854 3000 3000 93.5% chr4 - 11255347 11255495 149 browser details YourSeq 118 2864 3000 3000 94.2% chr3 - 125563091 125563236 146 browser details YourSeq 118 2858 3000 3000 91.2% chr15 - 60082971 60083111 141 browser details YourSeq 118 2869 3000 3000 93.9% chr5 + 65452060 65452190 131 browser details YourSeq 118 2857 3000 3000 91.4% chr5 + 33624494 33624636 143 browser details YourSeq 117 2872 3000 3000 93.8% chr4 - 140404624 140404751 128 browser details YourSeq 117 2864 3000 3000 93.4% chr4 - 133565232 133565374 143 Note: The 3000 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 3000 1 3000 3000 100.0% chr6 - 30502483 30505482 3000 browser details YourSeq 316 2407 2907 3000 90.7% chr9 + 106259410 106259776 367 browser details YourSeq 310 2407 2919 3000 90.3% chr10 - 7668844 7669209 366 browser details YourSeq 299 2407 2888 3000 95.5% chr3 + 103086637 103087126 490 browser details YourSeq 281 2414 2895 3000 89.1% chr8 - 26181555 26181881 327 browser details YourSeq 269 2407 2867 3000 95.4% chr4 - 73198453 73199033 581 browser details YourSeq 263 2223 2861 3000 95.3% chr11 - 101440396 101441113 718 browser details YourSeq 260 2164 2907 3000 88.7% chr12 + 110881043 110881452 410 browser details YourSeq 259 2407 2886 3000 90.8% chr7 - 19930973 19931298 326 browser details YourSeq 258 2407 2879 3000 88.6% chr19 + 4450202 4450513 312 browser details YourSeq 251 2142 2598 3000 94.0% chr11 + 115742585 116169946 427362 browser details YourSeq 241 2303 2857 3000 96.6% chrX - 8007270 8007862 593 browser details YourSeq 240 2164 2599 3000 95.5% chr7 - 12999955 13000434 480 browser details YourSeq 238 2407 2868 3000 90.9% chr3 + 144484206 144484608 403 browser details YourSeq 228 2413 2849 3000 89.7% chr19 + 8849775 8850104 330 browser details YourSeq 210 2200 2598 3000 97.4% chr10 - 62720847 62721269 423 browser details YourSeq 210 2407 2759 3000 92.5% chr1 - 151472110 151472320 211 browser details YourSeq 208 2202 2598 3000 96.1% chr10 + 128222889 128223506 618 browser details YourSeq 206 2407 2761 3000 98.6% chr11 - 85223087 85223763 677 browser details YourSeq 203 2407 2756 3000 91.9% chr8 - 125661843 125662075 233 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 http://www.alphaknockout.com/ Gene and protein information: Tmem209 transmembrane protein 209 [ Mus musculus (house mouse) ] Gene ID: 72649, updated on 12-Aug-2019 Gene summary Official Symbol Tmem209 provided by MGI Official Full Name transmembrane protein 209 provided by MGI Primary source MGI:MGI:1919899 See related Ensembl:ENSMUSG00000029782 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 AI428435; 2700094F01Rik Expression Ubiquitous expression in testis adult (RPKM 9.1), placenta adult (RPKM 7.6) and 28 other tissues See more Orthologs human all Genomic context Location: 6; 6 A3.3 See Tmem209 in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (30481022..30509787, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (30431233..30459706, complement) Chromosome 6 - NC_000072.6 Page 5 of 8 http://www.alphaknockout.com/ Transcript information: This gene has 11 transcripts Gene: Tmem209 ENSMUSG00000029782 Description transmembrane protein 209 [Source:MGI Symbol;Acc:MGI:1919899] Gene Synonyms 2700094F01Rik Location Chromosome 6: 30,479,053-30,509,783 reverse strand. GRCm38:CM000999.2 About this gene This gene has 11 transcripts (splice variants), 203 orthologues, is a member of 1 Ensembl protein family and is associated with 19 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tmem209- ENSMUST00000115160.9 3485 561aa ENSMUSP00000110813.3 Protein coding CCDS19972 Q8BRG8 TSL:1 204 GENCODE basic APPRIS P3 Tmem209- ENSMUST00000115157.7 2273 560aa ENSMUSP00000110810.1 Protein coding CCDS80511 Q8BRG8 TSL:1 203 GENCODE basic APPRIS ALT1 Tmem209- ENSMUST00000222934.1 1212 403aa ENSMUSP00000152560.1 Protein coding CCDS85026 Q8C214 TSL:5 211 GENCODE basic Tmem209- ENSMUST00000102991.8 1718 519aa ENSMUSP00000100056.2 Protein coding - F8WGT2 TSL:5 202 GENCODE basic Tmem209- ENSMUST00000064330.12 1679 438aa ENSMUSP00000067667.6 Protein coding - Q8BRG8 TSL:1 201 GENCODE basic Tmem209- ENSMUST00000154547.2 598 66aa ENSMUSP00000145248.1 Protein coding - A0A0N4SVU6 CDS 3' 209 incomplete TSL:2 Tmem209- ENSMUST00000148638.1 459 140aa ENSMUSP00000115567.1 Protein coding - D3YZT2 CDS 3' 206 incomplete TSL:2 Tmem209- ENSMUST00000151187.7 3998 403aa ENSMUSP00000138232.1 Nonsense mediated - Q8C214 TSL:1 208 decay Tmem209- ENSMUST00000138823.7 1905 561aa ENSMUSP00000138292.1 Nonsense mediated - Q8BRG8 TSL:5 205 decay Tmem209- ENSMUST00000150480.1 3128 No - Retained intron - - TSL:2 207 protein Tmem209- ENSMUST00000202269.1 1824 No - Retained intron - - TSL:NA 210 protein Page 6 of 8 http://www.alphaknockout.com/ 50.73 kb Forward strand 30.47Mb 30.48Mb 30.49Mb 30.50Mb 30.51Mb Genes (Comprehensive set from GENCODE M... Ssmem1-201 >protein coding Ssmem1-202 >protein coding Ssmem1-203 >protein coding Contigs < AC155848.6 Genes (Comprehensive set from GENCODE M... < Tmem209-208nonsense mediated decay < Tmem209-201protein coding < Tmem209-205nonsense mediated decay < Tmem209-204protein coding < Tmem209-203protein coding < Tmem209-202protein coding < Tmem209-210retained intron < Tmem209-209protein coding < Tmem209-207retained intron < Tmem209-206protein coding Regulatory Build 30.47Mb 30.48Mb 30.49Mb 30.50Mb 30.51Mb Reverse strand 50.73 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 http://www.alphaknockout.com/ Transcript: ENSMUST00000115160 < Tmem209-204protein coding Reverse strand 28.56 kb ENSMUSP000001108... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam Cytochrome B561-related PANTHER Cytochrome B561-related All sequence SNPs/in... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 561 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC, VectorBuilder. Page 8 of 8.
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