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Mouse Mapk8ip1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mapk8ip1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mapk8ip1 gene (NCBI Reference Sequence: NM_001202445 ; Ensembl: ENSMUSG00000027223 ) is located on Mouse chromosome 2. 12 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 12 (Transcript: ENSMUST00000111279). 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 Mapk8ip1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-316M14 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: Homozygous mutation of this gene results in a decreased susceptibility to ischemic brain injury.

Exon 2 starts from about 3.44% 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: 1015 bp, and the size of intron 2 for 3'-loxP site insertion: 1755 bp. The size of effective cKO region: ~606 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 3 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Mapk8ip1 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(7106bp) | A(21.17% 1504) | C(26.13% 1857) | T(22.19% 1577) | G(30.51% 2168)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr2 - 92391392 92394391 3000 browser details YourSeq 25 86 117 3000 88.9% chr5 - 125174726 125174756 31 browser details YourSeq 23 2105 2132 3000 80.8% chr12 + 57259816 57259841 26 browser details YourSeq 21 598 618 3000 100.0% chr17 - 69012436 69012456 21 browser details YourSeq 21 1835 1855 3000 100.0% chr14 - 71834831 71834851 21 browser details YourSeq 21 2633 2653 3000 100.0% chr13 - 18786326 18786346 21 browser details YourSeq 21 1929 1949 3000 100.0% chr9 + 13758696 13758716 21

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% chr2 - 92387786 92390785 3000 browser details YourSeq 36 1612 1648 3000 100.0% chrX + 93289005 93289086 82 browser details YourSeq 29 1620 1648 3000 100.0% chr6 - 21971135 21971163 29 browser details YourSeq 28 1620 1649 3000 96.7% chrX + 107589579 107589608 30 browser details YourSeq 26 1623 1648 3000 100.0% chr5 + 147258179 147258204 26 browser details YourSeq 23 1072 1094 3000 100.0% chr4 - 82528049 82528071 23 browser details YourSeq 23 1626 1648 3000 100.0% chr19 + 8820501 8820523 23 browser details YourSeq 22 1048 1069 3000 100.0% chr3 + 103340276 103340297 22 browser details YourSeq 20 659 678 3000 100.0% chr1 - 25096531 25096550 20

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: Mapk8ip1 mitogen-activated protein kinase 8 interacting protein 1 [ Mus musculus (house mouse) ] Gene ID: 19099, updated on 10-Oct-2019

Gene summary

Official Symbol Mapk8ip1 provided by MGI Official Full Name mitogen-activated protein kinase 8 interacting protein 1 provided by MGI Primary source MGI:MGI:1309464 See related Ensembl:ENSMUSG00000027223 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 IB1; Jip1; Skip; JIP-1; Prkm8ip; mjip-2a Expression Broad expression in cerebellum adult (RPKM 70.7), cortex adult (RPKM 62.2) and 17 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 E1 See Mapk8ip1 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (92383671..92401346, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (92223837..92241420, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 3 transcripts

Gene: Mapk8ip1 ENSMUSG00000027223

Description mitogen-activated protein kinase 8 interacting protein 1 [Source:MGI Symbol;Acc:MGI:1309464] Gene Synonyms IB1, JIP-1, Jip1, MAPK8IP1, Prkm8ip, Skip, mjip-2a Location Chromosome 2: 92,383,676-92,401,263 reverse strand. GRCm38:CM000995.2 About this gene This gene has 3 transcripts (splice variants), 205 orthologues, 10 paralogues, is a member of 1 Ensembl protein family and is associated with 14 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mapk8ip1-202 ENSMUST00000111279.8 3274 698aa ENSMUSP00000106910.2 Protein coding CCDS57180 Q9WVI9 TSL:1 GENCODE basic APPRIS ALT2

Mapk8ip1-201 ENSMUST00000050312.2 2930 707aa ENSMUSP00000050773.2 Protein coding CCDS16446 Q9WVI9 TSL:1 GENCODE basic APPRIS P3

Mapk8ip1-203 ENSMUST00000149653.1 1816 No protein - lncRNA - - TSL:1

37.59 kb Forward strand 92.38Mb 92.39Mb 92.40Mb 92.41Mb Genes Pex16-204 >lncRNA 1700029I15Rik-203 >retained intron D930015M05Rik-201 >lncRNA (Comprehensive set...

Pex16-201 >protein coding 1700029I15Rik-202 >protein coding

Pex16-203 >lncRNA 1700029I15Rik-201 >protein coding

Pex16-202 >lncRNA 1700029I15Rik-204 >nonsense mediated decay

Contigs AL731709.19 > Genes < Large2-211protein coding < Mapk8ip1-202protein coding < Cry2-201protein coding (Comprehensive set...

< Mapk8ip1-201protein coding < Cry2-202protein coding

< Mir7000-201miRNA

< Mapk8ip1-203lncRNA

Regulatory Build

92.38Mb 92.39Mb 92.40Mb 92.41Mb Reverse strand 37.59 kb

Regulation Legend

CTCF 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

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Transcript: ENSMUST00000111279

< Mapk8ip1-202protein coding

Reverse strand 9.06 kb

ENSMUSP00000106... MobiDB lite Low complexity (Seg) Superfamily SH3-like domain superfamily SSF50729

SMART SH3 domain PTB/PI domain

Pfam SH3 domain PTB/PI domain

PROSITE profiles SH3 domain PTB/PI domain

PANTHER PTHR47437

PTHR47437:SF3 Gene3D 2.30.30.40 PH-like domain superfamily

CDD JIP1, SH3 domain

cd01212

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 698

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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