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Mouse Map2k7 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Map2k7 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Map2k7 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Map2k7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Map2k7 gene (NCBI Reference Sequence: NM_001164172 ; Ensembl: ENSMUSG00000002948 ) is located on Mouse chromosome 8. 12 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 12 (Transcript: ENSMUST00000062686). Exon 3~12 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Map2k7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-463K24 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: Mice homozygous for disruptions in this gene die during embryogenesis. Exon 3~12 covers 86.82% of the coding region. Start codon is in exon 1, and stop codon is in exon 12. The size of intron 2 for 5'-loxP site insertion: 2568 bp. The size of effective cKO region: ~2836 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA T region A Wildtype allele G 5' gRNA region 3' 3 4 5 6 7 8 9 10 11 1 3 4 5 6 7 8 9 10 11 12 12 Targeting vector T A G Targeted allele T A G Constitutive KO allele (After Cre recombination) Legends Exon of mouse Map2k7 Homology arm cKO region Exon of mouse Gm49320 loxP site Page 2 of 8 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. 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(9023bp) | A(20.56% 1855) | C(27.09% 2444) | T(25.52% 2303) | G(26.83% 2421) 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. Page 3 of 8 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% chr8 + 4240055 4243054 3000 browser details YourSeq 37 2430 2514 3000 75.7% chr11 + 29867674 29867744 71 browser details YourSeq 36 1738 1775 3000 97.4% chr19 - 15751490 15751527 38 browser details YourSeq 34 770 807 3000 97.4% chr10 + 124960743 124960937 195 browser details YourSeq 32 1738 1769 3000 100.0% chr18 - 57998390 57998421 32 browser details YourSeq 32 1738 1769 3000 100.0% chr11 - 111829240 111829271 32 browser details YourSeq 32 1738 1769 3000 100.0% chr1 - 158728260 158728291 32 browser details YourSeq 31 519 620 3000 97.1% chr19 - 7052393 7052496 104 browser details YourSeq 31 1484 1520 3000 94.3% chr1 + 155226818 155226855 38 browser details YourSeq 29 1738 1766 3000 100.0% chr2 + 32738611 32738639 29 browser details YourSeq 29 1738 1768 3000 96.8% chr15 + 14792926 14792956 31 browser details YourSeq 27 1738 1764 3000 100.0% chr10 + 89606126 89606152 27 browser details YourSeq 26 780 810 3000 93.4% chr3 - 41500378 41500410 33 browser details YourSeq 25 1484 1521 3000 84.3% chr11 + 109857541 109857593 53 browser details YourSeq 20 1537 1556 3000 100.0% chr6 - 78078608 78078627 20 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% chr8 + 4245828 4248827 3000 browser details YourSeq 33 1770 1804 3000 91.2% chr16 + 20937133 20937166 34 browser details YourSeq 27 1773 1799 3000 100.0% chr4 - 148767061 148767087 27 browser details YourSeq 25 611 638 3000 84.7% chr3 - 132172291 132172316 26 browser details YourSeq 23 614 639 3000 96.0% chr1 + 193403911 193403938 28 browser details YourSeq 22 464 489 3000 92.4% chr15 - 64364696 64364721 26 browser details YourSeq 20 687 706 3000 100.0% chr12 + 41516235 41516254 20 Note: The 3000 bp section downstream of Exon 12 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Map2k7 mitogen-activated protein kinase kinase 7 [ Mus musculus (house mouse) ] Gene ID: 26400, updated on 10-Oct-2019 Gene summary Official Symbol Map2k7 provided by MGI Official Full Name mitogen-activated protein kinase kinase 7 provided by MGI Primary source MGI:MGI:1346871 See related Ensembl:ENSMUSG00000002948 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 Mek7; Mkk7; sek2; Jnkk2; MEK 7; JNKK 2; Mapkk7; Prkmk7; MAPKK 7; 5930412N11Rik Expression Ubiquitous expression in testis adult (RPKM 34.8), adrenal adult (RPKM 26.2) and 28 other tissues See more Orthologs human all Genomic context Location: 8; 8 A1.1 See Map2k7 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (4238740..4247897) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (4238740..4247897) Chromosome 8 - NC_000074.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 10 transcripts Gene: Map2k7 ENSMUSG00000002948 Description mitogen-activated protein kinase kinase 7 [Source:MGI Symbol;Acc:MGI:1346871] Gene Synonyms 5930412N11Rik, Jnkk2, MAP kinase kinase 7, MKK7, Prkmk7, sek2 Location Chromosome 8: 4,238,740-4,247,897 forward strand. GRCm38:CM001001.2 About this gene This gene has 10 transcripts (splice variants), 177 orthologues, 9 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Map2k7-202 ENSMUST00000062686.10 3525 435aa ENSMUSP00000054512.4 Protein coding CCDS52474 Q8CE90 TSL:1 GENCODE basic APPRIS ALT1 Map2k7-206 ENSMUST00000110998.8 3497 419aa ENSMUSP00000106626.2 Protein coding CCDS40209 Q8CE90 TSL:1 GENCODE basic APPRIS P3 Map2k7-201 ENSMUST00000003027.13 1626 468aa ENSMUSP00000003027.7 Protein coding CCDS40208 Q8CE90 TSL:1 GENCODE basic Map2k7-203 ENSMUST00000110994.8 1600 346aa ENSMUSP00000106622.1 Protein coding CCDS80854 Q8CE90 TSL:1 GENCODE basic Map2k7-207 ENSMUST00000110999.7 1578 452aa ENSMUSP00000106627.1 Protein coding CCDS80852 Q8CE90 TSL:1 GENCODE basic Map2k7-204 ENSMUST00000110995.7 1558 379aa ENSMUSP00000106623.1 Protein coding CCDS80853 Q8CE90 TSL:1 GENCODE basic Map2k7-205 ENSMUST00000110996.1 1188 391aa ENSMUSP00000106624.1 Protein coding - Q8CE90 TSL:1 GENCODE basic Map2k7-209 ENSMUST00000129866.7 760 79aa ENSMUSP00000146485.1 Protein coding - A0A140LHN8 CDS 5' incomplete TSL:3 Map2k7-208 ENSMUST00000129537.1 934 No protein - Retained intron - - TSL:2 Map2k7-210 ENSMUST00000207247.1 324 No protein - lncRNA - - TSL:2 Page 6 of 8 https://www.alphaknockout.com 29.16 kb Forward strand 4.23Mb 4.24Mb 4.25Mb Genes (Comprehensive set... Lrrc8e-201 >protein coding Map2k7-206 >protein coding Tgfbr3l-201 >protein coding Snapc2-201 >protein coding Lrrc8e-202 >protein coding Map2k7-202 >protein coding Tgfbr3l-202 >retained intronSnapc2-203 >protein coding Map2k7-207 >protein coding Tgfbr3l-205 >retained intron Snapc2-204 >protein coding Map2k7-201 >protein coding Tgfbr3l-203 >retained intron Gm49320-201 >nonsense mediated decay Snapc2-202 >protein coding Map2k7-204 >protein coding Tgfbr3l-204 >lncRNA Map2k7-203 >protein coding Map2k7-210 >lncRNA Map2k7-205 >protein coding Map2k7-209 >protein coding Map2k7-208 >retained intron Contigs AC123029.3 > Genes < Ctxn1-201protein coding (Comprehensive set... Regulatory Build 4.23Mb 4.24Mb 4.25Mb Reverse strand 29.16 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000062686 9.14 kb Forward strand Map2k7-202 >protein coding ENSMUSP00000054... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Protein kinase-like domain superfamily SMART Protein kinase domain Pfam Protein kinase domain PROSITE profiles Protein kinase domain PROSITE patterns Serine/threonine-protein kinase, active site PANTHER PTHR47238 PTHR47238:SF2 Gene3D 3.30.200.20 1.10.510.10 CDD cd06618 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 435 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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