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Mouse Mapk1ip1l Conditional Knockout Project (CRISPR/Cas9)

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Mouse Mapk1ip1l Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Mapk1ip1l Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Mapk1ip1l conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mapk1ip1l gene (NCBI Reference Sequence: NM_178684 ; Ensembl: ENSMUSG00000021840 ) is located on Mouse chromosome 14. 4 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000166743). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mapk1ip1l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-354G11 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 3 is not frameshift exon, and covers 96.28% of the coding region. The size of intron 2 for 5'-loxP site insertion: 1826 bp, and the size of intron 3 for 3'-loxP site insertion: 8298 bp. The size of effective cKO region: ~1199 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 4 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Mapk1ip1l Homology arm cKO region loxP site Page 2 of 7 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7699bp) | A(28.11% 2164) | C(21.33% 1642) | T(29.04% 2236) | G(21.52% 1657) 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 7 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% chr14 + 47307166 47310165 3000 browser details YourSeq 174 947 1256 3000 91.9% chr11 + 6497081 6567634 70554 browser details YourSeq 171 962 1256 3000 88.7% chr7 - 14617538 14617844 307 browser details YourSeq 165 948 1256 3000 95.2% chr4 - 148565536 148621530 55995 browser details YourSeq 161 949 1256 3000 94.6% chr1 - 160999085 160999459 375 browser details YourSeq 161 947 1138 3000 89.9% chr5 + 23606702 23606881 180 browser details YourSeq 159 947 1120 3000 96.0% chr7 - 141452702 141452877 176 browser details YourSeq 158 941 1121 3000 94.5% chr6 - 55528264 55528454 191 browser details YourSeq 156 950 1450 3000 86.4% chr3 - 68211073 68211261 189 browser details YourSeq 155 957 1460 3000 84.6% chr1 - 14614342 14614539 198 browser details YourSeq 154 950 1253 3000 93.3% chr9 + 64163384 64164011 628 browser details YourSeq 153 951 1137 3000 94.3% chr4 + 134986770 134986957 188 browser details YourSeq 153 947 1116 3000 95.3% chr14 + 119031022 119031192 171 browser details YourSeq 153 950 1111 3000 97.6% chr12 + 107385856 107386018 163 browser details YourSeq 151 949 1113 3000 96.4% chr1 - 88460870 88461038 169 browser details YourSeq 151 949 1111 3000 96.4% chr11 + 61753727 61753889 163 browser details YourSeq 150 947 1110 3000 94.5% chr10 - 91204805 91204967 163 browser details YourSeq 150 950 1111 3000 96.9% chr10 + 41794603 41794768 166 browser details YourSeq 149 947 1110 3000 95.8% chr6 - 17874852 17875020 169 browser details YourSeq 149 956 1400 3000 86.4% chr15 - 8377684 8378041 358 Note: The 3000 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 3000 1 3000 3000 100.0% chr14 + 47311365 47314364 3000 browser details YourSeq 298 1994 2762 3000 79.5% chr9 + 49332347 49333086 740 browser details YourSeq 260 469 797 3000 95.5% chr14 + 47314664 47314996 333 browser details YourSeq 171 573 2999 3000 91.0% chr1 + 152819176 153327264 508089 browser details YourSeq 142 447 836 3000 85.8% chr11 - 94694502 94694948 447 browser details YourSeq 132 2343 2760 3000 81.8% chr2 - 152469541 152469976 436 browser details YourSeq 124 573 810 3000 90.3% chr1 - 185013215 185147872 134658 browser details YourSeq 121 475 831 3000 87.9% chr1 + 128431494 128431894 401 browser details YourSeq 118 469 802 3000 84.2% chr3 - 79304187 79304500 314 browser details YourSeq 115 2801 3000 3000 91.4% chr18 + 35055629 35056257 629 browser details YourSeq 114 529 807 3000 88.6% chr15 - 77747826 77748104 279 browser details YourSeq 114 2837 3000 3000 84.8% chr6 + 113089521 113089684 164 browser details YourSeq 114 2477 3000 3000 74.4% chr10 + 76252869 76253055 187 browser details YourSeq 113 475 810 3000 93.2% chr11 - 83379654 83380010 357 browser details YourSeq 113 469 802 3000 68.4% chrX + 105944178 105944515 338 browser details YourSeq 112 544 813 3000 90.0% chr5 + 107528582 107528880 299 browser details YourSeq 110 544 801 3000 74.8% chr15 - 29527519 29527778 260 browser details YourSeq 108 566 836 3000 89.2% chr5 + 67637548 67637852 305 browser details YourSeq 108 2845 3000 3000 83.3% chr1 + 151464184 151464338 155 browser details YourSeq 102 2848 3000 3000 83.7% chr17 - 26151509 26151662 154 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Mapk1ip1l mitogen-activated protein kinase 1 interacting protein 1-like [ Mus musculus (house mouse) ] Gene ID: 218975, updated on 12-Aug-2019 Gene summary Official Symbol Mapk1ip1l provided by MGI Official Full Name mitogen-activated protein kinase 1 interacting protein 1-like provided by MGI Primary source MGI:MGI:2444022 See related Ensembl:ENSMUSG00000021840 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 9130202B12; C130032J12Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 12.6), limb E14.5 (RPKM 11.9) and 28 other tissues See more Orthologs human all Genomic context Location: 14; 14 C1 See Mapk1ip1l in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (47298274..47323206) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (47917989..47942766) Chromosome 14 - NC_000080.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Mapk1ip1l ENSMUSG00000021840 Description mitogen-activated protein kinase 1 interacting protein 1-like [Source:MGI Symbol;Acc:MGI:2444022] Gene Synonyms C130032J12Rik Location Chromosome 14: 47,298,266-47,323,204 forward strand. GRCm38:CM001007.2 About this gene This gene has 5 transcripts (splice variants), 133 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mapk1ip1l- ENSMUST00000166743.8 4542 242aa ENSMUSP00000127475.1 Protein coding CCDS49469 Q8BH93 TSL:1 202 GENCODE basic APPRIS P1 Mapk1ip1l- ENSMUST00000164235.2 4531 242aa ENSMUSP00000132875.1 Protein coding CCDS49469 Q8BH93 TSL:1 201 GENCODE basic APPRIS P1 Mapk1ip1l- ENSMUST00000228058.1 840 153aa ENSMUSP00000154193.1 Protein coding - A0A2I3BQG9 CDS 5' 205 incomplete Mapk1ip1l- ENSMUST00000227554.1 584 139aa ENSMUSP00000154689.1 Protein coding - A0A2I3BRS9 CDS 3' 203 incomplete Mapk1ip1l- ENSMUST00000227941.1 3105 No - Retained - - - 204 protein intron 44.94 kb Forward strand 47.29Mb 47.30Mb 47.31Mb 47.32Mb 47.33Mb Genes (Comprehensive set... Socs4-201 >protein coding Mapk1ip1l-204 >retained intron Mapk1ip1l-201 >protein coding Socs4-202 >protein coding Mapk1ip1l-202 >protein coding Mapk1ip1l-203 >protein coding Mapk1ip1l-205 >protein coding Contigs < AC156016.2 Genes < Gm9502-201TEC < Gm49190-201processed pseudogene (Comprehensive set... < Gm49189-201lncRNA Regulatory Build 47.29Mb 47.30Mb 47.31Mb 47.32Mb 47.33Mb Reverse strand 44.94 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 pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000166743 24.83 kb Forward strand Mapk1ip1l-202 >protein coding ENSMUSP00000127... MobiDB lite Low complexity (Seg) Pfam MAPK-interacting and spindle-stabilising protein PANTHER PTHR35973:SF1 MAPK-interacting and spindle-stabilising protein All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 242 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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