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Mouse Ppm1b Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ppm1b Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ppm1b Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ppm1b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ppm1b gene (NCBI Reference Sequence: NM_011151 ; Ensembl: ENSMUSG00000061130 ) is located on Mouse chromosome 17. 6 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 6 (Transcript: ENSMUST00000112304). 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 Ppm1b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-439E12 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 KO results in early pre-implantation lethality. A hypomorphic mutation results in increased sensitivityto Tnf-induced necroptosis and early death. 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: 35285 bp, and the size of intron 2 for 3'-loxP site insertion: 8726 bp. The size of effective cKO region: ~1346 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ppm1b Homology arm cKO region 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. 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(7846bp) | A(27.63% 2168) | C(19.67% 1543) | T(30.05% 2358) | G(22.65% 1777) 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 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% chr17 + 84990445 84993444 3000 browser details YourSeq 300 725 3000 3000 91.8% chr4 + 131736911 131999380 262470 browser details YourSeq 103 2465 2996 3000 76.7% chr17 - 83293585 83293966 382 browser details YourSeq 95 919 1057 3000 91.4% chr14 - 31085823 31085964 142 browser details YourSeq 88 947 1059 3000 91.3% chrX + 139633722 139633832 111 browser details YourSeq 88 923 1044 3000 88.6% chr10 + 81630542 81630665 124 browser details YourSeq 87 2669 2968 3000 95.9% chr4 + 135893838 135894268 431 browser details YourSeq 84 703 998 3000 88.2% chr5 + 148427711 148428112 402 browser details YourSeq 82 2669 2965 3000 79.8% chr2 - 144563946 144564176 231 browser details YourSeq 82 695 1021 3000 90.2% chr17 - 70868012 70868377 366 browser details YourSeq 82 2509 2994 3000 75.3% chr15 - 96292281 96292559 279 browser details YourSeq 82 2891 3000 3000 89.6% chr12 + 102298612 102298723 112 browser details YourSeq 82 2727 3000 3000 80.2% chr11 + 94583943 94584171 229 browser details YourSeq 79 2891 3000 3000 88.5% chr6 - 85936345 85936456 112 browser details YourSeq 79 2727 2966 3000 93.6% chr2 - 154626266 154626600 335 browser details YourSeq 79 2892 3000 3000 87.2% chr11 - 34894067 34894177 111 browser details YourSeq 79 938 1044 3000 93.5% chr1 - 181875096 181875206 111 browser details YourSeq 77 2727 2983 3000 88.9% chr3 - 89500206 89500488 283 browser details YourSeq 77 2891 2996 3000 86.7% chr5 + 8506440 8506545 106 browser details YourSeq 76 938 1038 3000 92.4% chr5 + 98752312 98752419 108 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% chr17 + 84994791 84997790 3000 browser details YourSeq 70 2911 2995 3000 93.0% chr10 - 70966057 70966154 98 browser details YourSeq 69 2900 2983 3000 92.8% chr11 + 44479467 44479563 97 browser details YourSeq 63 2911 2995 3000 93.4% chr10 - 122595954 122596048 95 browser details YourSeq 61 2911 3000 3000 91.8% chr10 - 96155466 96155763 298 browser details YourSeq 61 2927 2996 3000 95.8% chr13 + 90554633 90555021 389 browser details YourSeq 58 2923 3000 3000 92.9% chr1 - 153706142 153706232 91 browser details YourSeq 55 2932 2996 3000 93.9% chr5 - 104846709 104846776 68 browser details YourSeq 55 2900 2999 3000 89.9% chr1 + 171752445 171752557 113 browser details YourSeq 53 2934 2995 3000 95.2% chr1 - 183237561 183237627 67 browser details YourSeq 53 2942 3000 3000 96.7% chr18 + 11766379 11766441 63 browser details YourSeq 52 2934 2994 3000 93.4% chr15 - 35789006 35789068 63 browser details YourSeq 52 2934 2996 3000 93.7% chr10 - 115374666 115374732 67 browser details YourSeq 52 2941 3000 3000 95.0% chr10 - 41629200 41629262 63 browser details YourSeq 52 2941 2996 3000 98.3% chr6 + 136508436 136508494 59 browser details YourSeq 52 2934 2994 3000 95.0% chr13 + 38859480 38859545 66 browser details YourSeq 52 2944 3000 3000 98.3% chr12 + 98606688 98607002 315 browser details YourSeq 51 2944 3000 3000 96.5% chr3 + 122640246 122640305 60 browser details YourSeq 51 2944 3000 3000 96.5% chr14 + 58155500 58155559 60 browser details YourSeq 51 2944 2996 3000 100.0% chr1 + 16549774 16631296 81523 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Ppm1b protein phosphatase 1B, magnesium dependent, beta isoform [ Mus musculus (house mouse) ] Gene ID: 19043, updated on 10-Oct-2019 Gene summary Official Symbol Ppm1b provided by MGI Official Full Name protein phosphatase 1B, magnesium dependent, beta isoform provided by MGI Primary source MGI:MGI:101841 See related Ensembl:ENSMUSG00000061130 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 PP2CB Expression Ubiquitous expression in testis adult (RPKM 20.4), cerebellum adult (RPKM 16.6) and 28 other tissues See more Orthologs human all Genomic context Location: 17 E4; 17 55.13 cM See Ppm1b in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (84956741..85023992) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (85356081..85423332) Chromosome 17 - NC_000083.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Ppm1b ENSMUSG00000061130 Description protein phosphatase 1B, magnesium dependent, beta isoform [Source:MGI Symbol;Acc:MGI:101841] Gene Synonyms PP2CB Location Chromosome 17: 84,956,741-85,023,991 forward strand. GRCm38:CM001010.2 About this gene This gene has 8 transcripts (splice variants), 254 orthologues, 16 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ppm1b- ENSMUST00000112304.9 3323 390aa ENSMUSP00000107923.2 Protein coding CCDS37710 P36993 Q546R1 TSL:1 202 GENCODE basic APPRIS P3 Ppm1b- ENSMUST00000080217.13 2670 477aa ENSMUSP00000079107.6 Protein coding CCDS50199 Q99NF7 TSL:1 201 GENCODE basic APPRIS ALT2 Ppm1b- ENSMUST00000234540.1 1633 393aa ENSMUSP00000157282.1 Protein coding CCDS50200 P36993 GENCODE basic 206 APPRIS ALT2 Ppm1b- ENSMUST00000112305.9 1591 390aa ENSMUSP00000107924.3 Protein coding CCDS50201 P36993 TSL:1 203 GENCODE basic APPRIS ALT2 Ppm1b- ENSMUST00000112307.3 1499 393aa ENSMUSP00000107926.3 Protein coding CCDS50200 P36993 TSL:1 204 GENCODE basic APPRIS ALT2 Ppm1b- ENSMUST00000234332.1 1290 390aa ENSMUSP00000157013.1 Protein coding CCDS50201 P36993 GENCODE basic 205 APPRIS ALT2 Ppm1b- ENSMUST00000234851.1 1307 402aa ENSMUSP00000157058.1 Protein coding - P36993 GENCODE basic 208 APPRIS ALT2 Ppm1b- ENSMUST00000234710.1 1320 No - Retained - - - 207 protein intron Page 6 of 8 https://www.alphaknockout.com 87.25 kb Forward strand 84.96Mb 84.98Mb 85.00Mb 85.02Mb Genes (Comprehensive set... Ppm1b-203 >protein coding Ppm1b-201 >protein coding Slc3a1-201 >protein coding Ppm1b-202 >protein coding Ppm1b-205 >protein coding Ppm1b-207 >retained intron Ppm1b-206 >protein coding Ppm1b-204 >protein coding Ppm1b-208 >protein coding Contigs < AC142103.3 Genes < 1110020A21Rik-202lncRNA (Comprehensive set... < 1110020A21Rik-203lncRNA < 1110020A21Rik-201lncRNA Regulatory Build 84.96Mb 84.98Mb 85.00Mb 85.02Mb Reverse strand 87.25 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding
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