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Mouse Il1rapl2 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Il1rapl2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Il1rapl2 gene (NCBI Reference Sequence: NM_030688 ; Ensembl: ENSMUSG00000059203 ) is located on Mouse chromosome X. 11 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 11 (Transcript: ENSMUST00000113063). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Il1rapl2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-210H6 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 2 starts from about 100% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 80058 bp, and the size of intron 2 for 3'-loxP site insertion: 522727 bp. The size of effective cKO region: ~582 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 2 11 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Il1rapl2 Homology arm cKO region loxP site
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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(7082bp) | A(30.37% 2151) | C(17.16% 1215) | T(33.76% 2391) | G(18.71% 1325)
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.
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BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chrX + 137648471 137651470 3000 browser details YourSeq 235 438 698 3000 95.5% chr3 + 31787690 31787952 263 browser details YourSeq 220 438 694 3000 93.4% chr18 - 18180909 18181171 263 browser details YourSeq 218 438 677 3000 95.9% chr5 + 97919277 97919518 242 browser details YourSeq 216 438 680 3000 94.7% chrX + 161437700 161437943 244 browser details YourSeq 214 438 700 3000 92.8% chr10 - 35909044 35909296 253 browser details YourSeq 212 438 674 3000 95.3% chr9 + 105016271 105016516 246 browser details YourSeq 211 438 668 3000 96.2% chr14 - 70799329 70799567 239 browser details YourSeq 209 438 677 3000 94.1% chr8 - 40790843 40791082 240 browser details YourSeq 209 438 674 3000 94.5% chrX + 150518853 150519092 240 browser details YourSeq 207 340 658 3000 93.7% chrX - 137470169 137470763 595 browser details YourSeq 206 438 686 3000 92.6% chr17 - 85049301 85049552 252 browser details YourSeq 205 441 674 3000 95.2% chr2 + 5537546 5537781 236 browser details YourSeq 205 438 668 3000 94.8% chr13 + 64936333 64936567 235 browser details YourSeq 204 438 665 3000 94.8% chr19 - 54301918 54302145 228 browser details YourSeq 204 446 682 3000 93.6% chr3 + 78615499 78615737 239 browser details YourSeq 204 438 676 3000 92.9% chr14 + 62049161 62049400 240 browser details YourSeq 203 438 700 3000 89.1% chr15 - 60434012 60434256 245 browser details YourSeq 203 438 676 3000 93.2% chr18 + 51278236 51278475 240 browser details YourSeq 203 438 668 3000 94.4% chr15 + 29667947 29668180 234
Note: The 3000 bp section upstream of Exon 2 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% chrX + 137652053 137655052 3000 browser details YourSeq 32 2397 2450 3000 97.1% chr19 - 14848625 14848679 55 browser details YourSeq 30 2967 3000 3000 94.2% chr15 + 57979633 57979666 34 browser details YourSeq 28 1254 1301 3000 84.4% chr14 - 112987839 112987884 46 browser details YourSeq 25 2928 2955 3000 96.5% chr10 + 18809637 18809666 30 browser details YourSeq 25 96 126 3000 92.9% chr1 + 109625191 109625222 32 browser details YourSeq 23 2394 2419 3000 96.0% chr18 + 58280492 58280521 30 browser details YourSeq 22 2396 2419 3000 95.9% chr1 + 178882503 178882526 24
Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Il1rapl2 interleukin 1 receptor accessory protein-like 2 [ Mus musculus (house mouse) ] Gene ID: 60367, updated on 12-Aug-2019
Gene summary
Official Symbol Il1rapl2 provided by MGI Official Full Name interleukin 1 receptor accessory protein-like 2 provided by MGI Primary source MGI:MGI:1913106 See related Ensembl:ENSMUSG00000059203 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 Expression Low expression observed in reference dataset See more Orthologs human all
Genomic context
Location: X; X F1 See Il1rapl2 in Genome Data Viewer Exon count: 11
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (137570754..138849077)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (134106088..135381494)
Chromosome X - NC_000086.7
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Transcript information: This gene has 3 transcripts
Gene: Il1rapl2 ENSMUSG00000059203
Description interleukin 1 receptor accessory protein-like 2 [Source:MGI Symbol;Acc:MGI:1913106] Location Chromosome X: 137,570,608-138,846,946 forward strand. GRCm38:CM001013.2 About this gene This gene has 3 transcripts (splice variants), 199 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Il1rapl2- ENSMUST00000113063.7 3437 686aa ENSMUSP00000108686.1 Protein coding CCDS30429 Q0VBP3 TSL:1 202 Q9ERS6 GENCODE basic APPRIS P1
Il1rapl2- ENSMUST00000075471.3 2061 686aa ENSMUSP00000074917.3 Protein coding CCDS30429 Q0VBP3 TSL:5 201 Q9ERS6 GENCODE basic APPRIS P1
Il1rapl2- ENSMUST00000130879.1 2670 No - Retained - - TSL:5 203 protein intron
1.30 Mb Forward strand
137.6Mb 137.8Mb 138.0Mb 138.2Mb 138.4Mb 138.6Mb 138.8Mb Genes (Comprehensive set... Il1rapl2-202 >protein coding
Il1rapl2-203 >retained intron
Il1rapl2-201 >protein coding
Contigs AL691422.15 > BX890639.3 > AL672286.9 > AL672205.7 > BX572626.6 > AL672067.5 >
Genes < Gm14988-201processed pseudogene < Gm14989-201processed pseudogene < Gm23958-201snRNA < Gm15001-201processed pseudogene (Comprehensive set...
< Gm25393-201misc RNA < Gm24675-201misc R < Tex13a-201protein coding Regulatory Build 137.6Mb 137.8Mb 138.0Mb 138.2Mb 138.4Mb 138.6Mb 138.8Mb Reverse strand 1.30 Mb 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 pseudogene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000113063 1.28 Mb Forward strand Il1rapl2-202 >protein coding ENSMUSP00000108... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily Toll/interleukin-1 receptor homology (TIR) domain superfamily SMART Immunoglobulin subtype Toll/interleukin-1 receptor homology (TIR) domain Immunoglobulin subtype 2 Prints Interleukin-1 receptor type II PR01537 Pfam IL-18Ra-like, immunoglobulin domain Toll/interleukin-1 receptor homology (TIR) domain Immunoglobulin-like domain Immunoglobulin I-set PROSITE profiles Immunoglobulin-like domain Toll/interleukin-1 receptor homology (TIR) domain PANTHER PTHR11890:SF10 Interleukin-1 receptor family Gene3D Immunoglobulin-like fold Toll/interleukin-1 receptor homology (TIR) domain superfamily CDD cd05896 cd05757 cd00096 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 686 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7