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Mouse Map4k3 Knockout Project (CRISPR/Cas9)

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Mouse Map4k3 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Map4k3 Knockout Project (CRISPR/Cas9) Objective: To create a Map4k3 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Map4k3 gene (NCBI Reference Sequence: NM_001290345 ; Ensembl: ENSMUSG00000024242 ) is located on Mouse chromosome 17. 34 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 34 (Transcript: ENSMUST00000025089). 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 gene trap allele exhibit decreased susceptibility to experimental autoimmune encephalomyelitis, decreased stimulated immunoglobin production, decreased stimulated T cell proliferation, and abnormal Th1, Th2, and Th17 differentiation. Exon 3 starts from about 5.78% of the coding region. Exon 3~5 covers 7.9% of the coding region. The size of effective KO region: ~9671 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 3 4 5 34 Legends Exon of mouse Map4k3 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 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 502 bp section downstream of Exon 5 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(25.2% 504) | C(21.35% 427) | T(29.1% 582) | G(24.35% 487) 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(502bp) | A(25.9% 130) | C(18.13% 91) | T(36.25% 182) | G(19.72% 99) Note: The 502 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 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% chr17 - 80664083 80666082 2000 browser details YourSeq 29 1522 1558 2000 76.7% chr3 + 95773703 95773732 30 browser details YourSeq 26 1370 1400 2000 96.5% chrX - 12515861 12515898 38 browser details YourSeq 25 1524 1550 2000 88.5% chr5 - 67051217 67051242 26 browser details YourSeq 25 1521 1545 2000 100.0% chr1 - 15702071 15702095 25 browser details YourSeq 25 1123 1160 2000 82.8% chr8 + 69712561 69712596 36 browser details YourSeq 24 1523 1546 2000 100.0% chr1 + 57228800 57228823 24 browser details YourSeq 24 1521 1546 2000 96.2% chr1 + 20924518 20924543 26 browser details YourSeq 23 1523 1545 2000 100.0% chr3 - 49907236 49907258 23 browser details YourSeq 23 1521 1543 2000 100.0% chr14 + 16250074 16250096 23 browser details YourSeq 23 1520 1542 2000 100.0% chr1 + 104000964 104000986 23 browser details YourSeq 22 1523 1544 2000 100.0% chr6 - 134405216 134405237 22 browser details YourSeq 22 1523 1546 2000 95.9% chr1 + 65924810 65924833 24 browser details YourSeq 21 1478 1498 2000 100.0% chr15 - 102834906 102834926 21 browser details YourSeq 21 1523 1543 2000 100.0% chr14 - 60122981 60123001 21 browser details YourSeq 20 753 772 2000 100.0% chr1 + 51998677 51998696 20 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 502 1 502 502 100.0% chr17 - 80653910 80654411 502 browser details YourSeq 29 134 179 502 94.0% chr2 - 79604460 79604506 47 browser details YourSeq 27 138 176 502 96.6% chr10 - 119985872 119985911 40 browser details YourSeq 24 136 162 502 96.2% chr11 + 116704787 116704814 28 browser details YourSeq 23 145 180 502 83.4% chr7 + 133325306 133325342 37 browser details YourSeq 23 140 163 502 100.0% chr1 + 119533535 119533559 25 browser details YourSeq 23 354 377 502 100.0% chr1 + 57030985 57031011 27 browser details YourSeq 22 401 425 502 95.9% chr14 + 120037009 120037038 30 browser details YourSeq 21 474 494 502 100.0% chr16 - 38348151 38348171 21 browser details YourSeq 20 467 486 502 100.0% chr17 - 79263738 79263757 20 browser details YourSeq 20 472 491 502 100.0% chr12 - 89842755 89842774 20 browser details YourSeq 20 143 162 502 100.0% chr11 - 104008619 104008638 20 browser details YourSeq 20 136 155 502 100.0% chr11 - 80290936 80290955 20 browser details YourSeq 20 473 492 502 100.0% chr16 + 22183450 22183469 20 browser details YourSeq 20 476 495 502 100.0% chr1 + 85105462 85105481 20 browser details YourSeq 20 476 495 502 100.0% chr1 + 85265623 85265642 20 Note: The 502 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Map4k3 mitogen-activated protein kinase kinase kinase kinase 3 [ Mus musculus (house mouse) ] Gene ID: 225028, updated on 10-Oct-2019 Gene summary Official Symbol Map4k3 provided by MGI Official Full Name mitogen-activated protein kinase kinase kinase kinase 3 provided by MGI Primary source MGI:MGI:2154405 See related Ensembl:ENSMUSG00000024242 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 Glk; MEKKK3; MEKKK 3; MAPKKKK3; RAB8IPL1; 4833416M01Rik; 4833416M07Rik; 9530052P13Rik Expression Ubiquitous expression in bladder adult (RPKM 16.8), limb E14.5 (RPKM 14.8) and 28 other tissues See more Orthologs human all Genomic context Location: 17; 17 E3 See Map4k3 in Genome Data Viewer Exon count: 36 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (80580513..80728806, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (80979852..81127433, complement) Chromosome 17 - NC_000083.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Map4k3 ENSMUSG00000024242 Description mitogen-activated protein kinase kinase kinase kinase 3 [Source:MGI Symbol;Acc:MGI:2154405] Gene Synonyms 9530052P13Rik Location Chromosome 17: 80,580,512-80,728,485 reverse strand. GRCm38:CM001010.2 About this gene This gene has 8 transcripts (splice variants), 211 orthologues, 35 paralogues, is a member of 1 Ensembl protein family and is associated with 16 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Map4k3-201 ENSMUST00000025089.8 4220 894aa ENSMUSP00000025089.7 Protein coding CCDS70847 Q99JP0 TSL:5 GENCODE basic APPRIS P2 Map4k3-202 ENSMUST00000112389.8 4231 896aa ENSMUSP00000108008.2 Protein coding - E9QNE9 TSL:5 GENCODE basic APPRIS ALT1 Map4k3-203 ENSMUST00000234133.1 4088 873aa ENSMUSP00000157308.1 Protein coding - A0A3Q4EGQ9 GENCODE basic APPRIS ALT1 Map4k3-208 ENSMUST00000234585.1 3769 No protein - Retained intron - - - Map4k3-206 ENSMUST00000234481.1 3647 No protein - Retained intron - - - Map4k3-204 ENSMUST00000234200.1 2372 No protein - Retained intron - - - Map4k3-207 ENSMUST00000234486.1 561 No protein - Retained intron - - - Map4k3-205 ENSMUST00000234249.1 484 No protein - lncRNA - - - Page 7 of 9 https://www.alphaknockout.com 167.97 kb Forward strand 80.60Mb 80.65Mb 80.70Mb Genes Gm9959-201 >pseudogene (Comprehensive set... Contigs < AC131712.3 AC169501.2 > Genes (Comprehensive set... < Cdkl4-203protein coding < Map4k3-206retained intron < Map4k3-207retained intron < Cdkl4-202protein coding < Map4k3-205lncRNA < Map4k3-202protein coding < Map4k3-201protein coding < Map4k3-203protein coding < Map4k3-208retained intron < Map4k3-204retained intron Regulatory Build 80.60Mb 80.65Mb 80.70Mb Reverse strand 167.97 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript pseudogene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000025089 < Map4k3-201protein coding Reverse strand 147.58 kb ENSMUSP00000025... MobiDB lite Low complexity (Seg) Superfamily Protein kinase-like domain superfamily SMART Protein kinase domain Citron homology (CNH) domain Pfam Protein kinase domain Citron homology (CNH) domain PROSITE profiles Protein kinase domain Citron homology (CNH) domain PROSITE patterns Protein kinase, ATP binding site PIRSF Mitogen-activated protein (MAP) kinase kinase kinase kinase PANTHER PTHR24361:SF205 PTHR24361 Gene3D 1.10.510.10 CDD cd06613 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 800 894 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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