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Mouse Eif4e3 Knockout Project (CRISPR/Cas9)

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Mouse Eif4e3 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Eif4e3 Knockout Project (CRISPR/Cas9) Objective: To create a Eif4e3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Eif4e3 gene (NCBI Reference Sequence: NM_025829 ; Ensembl: ENSMUSG00000093661 ) is located on Mouse chromosome 6. 7 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000032151). Exon 3~5 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 knock-out allele exhibit decreased bone trabecula number. Exon 3 starts from about 32.05% of the coding region. Exon 3~5 covers 35.91% of the coding region. The size of effective KO region: ~4602 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 7 Legends Exon of mouse Eif4e3 Knockout region Page 2 of 8 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 3 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 2000 bp section downstream of Exon 5 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(29.65% 593) | C(19.05% 381) | T(31.05% 621) | G(20.25% 405) Note: The 2000 bp section upstream of Exon 3 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(2000bp) | A(22.7% 454) | C(22.45% 449) | T(31.9% 638) | G(22.95% 459) Note: The 2000 bp section downstream of Exon 5 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 8 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% chr6 - 99640711 99642710 2000 browser details YourSeq 81 359 472 2000 85.9% chr13 - 74221950 74222065 116 browser details YourSeq 81 326 473 2000 84.7% chr14 + 65109274 65109432 159 browser details YourSeq 79 1492 1651 2000 84.5% chr8 - 95882765 95882926 162 browser details YourSeq 78 1082 1224 2000 75.2% chr1 - 183746543 183746683 141 browser details YourSeq 77 346 459 2000 84.3% chr1 + 72523062 72523177 116 browser details YourSeq 76 361 473 2000 86.7% chr3 - 104536475 104536588 114 browser details YourSeq 75 369 1134 2000 91.4% chr10 + 39177605 39380592 202988 browser details YourSeq 74 1516 1689 2000 76.9% chr7 - 97462049 97462223 175 browser details YourSeq 74 365 467 2000 94.2% chr18 - 10978402 10978509 108 browser details YourSeq 70 359 473 2000 90.6% chr11 - 43625656 43625772 117 browser details YourSeq 70 308 424 2000 86.6% chr1 - 57646568 57646695 128 browser details YourSeq 70 375 473 2000 90.9% chr13 + 60537724 60537823 100 browser details YourSeq 69 370 467 2000 88.9% chr2 - 135862301 135862400 100 browser details YourSeq 66 371 459 2000 88.4% chr1 - 78617283 78914782 297500 browser details YourSeq 66 1103 1227 2000 80.7% chr6 + 35190176 35190298 123 browser details YourSeq 65 358 459 2000 88.3% chr12 - 83711828 83711934 107 browser details YourSeq 64 304 429 2000 95.8% chr13 + 70051987 70052142 156 browser details YourSeq 64 1516 1642 2000 80.7% chr1 + 177661176 177661301 126 browser details YourSeq 62 344 473 2000 84.0% chr14 + 70619524 70619652 129 Note: The 2000 bp section upstream of Exon 3 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 2000 1 2000 2000 100.0% chr6 - 99634109 99636108 2000 browser details YourSeq 77 1204 1316 2000 87.4% chr12 - 119867477 119867588 112 browser details YourSeq 52 1239 1305 2000 79.0% chr3 + 40423735 40423791 57 browser details YourSeq 46 1259 1314 2000 90.4% chr1 + 186749675 186749729 55 browser details YourSeq 43 1230 1300 2000 75.0% chr1 - 193576728 193576776 49 browser details YourSeq 36 1230 1314 2000 92.9% chr10 - 120485219 120485306 88 browser details YourSeq 34 1258 1297 2000 94.6% chr3 + 40423735 40423791 57 browser details YourSeq 31 1220 1251 2000 100.0% chr19 + 28218824 28218865 42 browser details YourSeq 30 1228 1292 2000 65.7% chr19 + 28218824 28218865 42 browser details YourSeq 29 1255 1284 2000 100.0% chr19 + 28218824 28218865 42 browser details YourSeq 28 1221 1255 2000 80.7% chr1 + 188934241 188934272 32 browser details YourSeq 27 1262 1292 2000 96.6% chr13 - 106811044 106811076 33 browser details YourSeq 27 1203 1248 2000 64.3% chr2 + 128379545 128379572 28 browser details YourSeq 26 1264 1295 2000 75.0% chr12 - 73111219 73111246 28 browser details YourSeq 23 1279 1306 2000 80.8% chr15 + 69867461 69867486 26 browser details YourSeq 22 600 621 2000 100.0% chr3 + 81582766 81582787 22 Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Eif4e3 eukaryotic translation initiation factor 4E member 3 [ Mus musculus (house mouse) ] Gene ID: 66892, updated on 12-Aug-2019 Gene summary Official Symbol Eif4e3 provided by MGI Official Full Name eukaryotic translation initiation factor 4E member 3 provided by MGI Primary source MGI:MGI:1914142 See related Ensembl:ENSMUSG00000093661 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 eIF4E-3; AI451927; 1300018P11Rik Expression Ubiquitous expression in bladder adult (RPKM 17.2), subcutaneous fat pad adult (RPKM 13.9) and 27 other tissues See Orthologs more human all Genomic context Location: 6; 6 D3 See Eif4e3 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (99621879..99692873, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (99575131..99616765, complement) Chromosome 6 - NC_000072.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Eif4e3 ENSMUSG00000093661 Description eukaryotic translation initiation factor 4E member 3 [Source:MGI Symbol;Acc:MGI:1914142] Gene Synonyms 1300018P11Rik, eIF4E-3 Location Chromosome 6: 99,625,135-99,666,771 reverse strand. GRCm38:CM000999.2 About this gene This gene has 1 transcript (splice variant), 259 orthologues, 3 paralogues, 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 Eif4e3-201 ENSMUST00000032151.2 2515 207aa ENSMUSP00000032151.2 Protein coding CCDS20386 Q9DBB5 TSL:1 GENCODE basic APPRIS P1 61.64 kb Forward strand 99.62Mb 99.63Mb 99.64Mb 99.65Mb 99.66Mb 99.67Mb Contigs AC152987.1 > < AC122929.4 Genes < Gm20696-201nonsense mediated decay (Comprehensive set... < Gm20696-203retained intron < Gm44104-201TEC < Eif4e3-201protein coding Regulatory Build 99.62Mb 99.63Mb 99.64Mb 99.65Mb 99.66Mb 99.67Mb Reverse strand 61.64 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000032151 < Eif4e3-201protein coding Reverse strand 41.64 kb ENSMUSP00000032... PDB-ENSP mappings Low complexity (Seg) Superfamily Translation Initiation factor eIF- 4e-like Pfam Translation Initiation factor eIF- 4e PANTHER PTHR11960:SF18 Translation Initiation factor eIF- 4e Gene3D Translation Initiation factor eIF- 4e-like All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 207 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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