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Mouse Pgam5 Conditional Knockout Project (CRISPR/Cas9)

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https://www.alphaknockout.com Mouse Pgam5 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pgam5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pgam5 gene (NCBI Reference Sequence: NM_001163538 ; Ensembl: ENSMUSG00000029500 ) is located on Mouse chromosome 5. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000112505). 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 Pgam5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-24E20 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: Homozygotes for a null allele show Parkinson-like movement, dopaminergic neuron loss, and altered mitochondrial homeostasis. Homozygotes for a different null allele are smaller, with males born at sub-Mendelian ratios. Macrophages show impaired inflammasome activation and IL-1beta secretion. Exon 2 starts from about 21.88% 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: 2386 bp, and the size of intron 2 for 3'-loxP site insertion: 1050 bp. The size of effective cKO region: ~679 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 5 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pgam5 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. 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(7179bp) | A(24.6% 1766) | C(23.42% 1681) | T(27.75% 1992) | G(24.24% 1740) 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 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% chr5 - 110267516 110270515 3000 browser details YourSeq 134 674 865 3000 84.9% chr12 + 91482050 91482241 192 browser details YourSeq 125 368 1378 3000 94.3% chr14 - 59355258 59442524 87267 browser details YourSeq 109 368 489 3000 95.1% chr5 + 8128727 8128850 124 browser details YourSeq 107 375 490 3000 96.5% chr4 - 99427456 99427572 117 browser details YourSeq 105 375 489 3000 93.0% chr4 + 59276854 59276966 113 browser details YourSeq 102 368 477 3000 96.4% chr10 + 29972289 29972398 110 browser details YourSeq 101 368 486 3000 94.7% chr8 - 59264570 59264693 124 browser details YourSeq 101 368 472 3000 98.1% chr11 + 55068757 55068861 105 browser details YourSeq 100 367 477 3000 95.5% chr12 - 21578680 21578791 112 browser details YourSeq 99 370 477 3000 96.3% chrX - 51146886 51146997 112 browser details YourSeq 99 368 482 3000 93.1% chr17 + 28976584 28976698 115 browser details YourSeq 97 370 477 3000 95.4% chr2 - 154489976 154490093 118 browser details YourSeq 97 375 482 3000 95.4% chr8 + 78479266 78479375 110 browser details YourSeq 97 375 477 3000 97.1% chr2 + 163646439 163646541 103 browser details YourSeq 96 368 477 3000 93.7% chr2 - 49453656 49453765 110 browser details YourSeq 94 375 477 3000 96.1% chr8 - 45007323 45007426 104 browser details YourSeq 93 368 466 3000 97.0% chr16 - 79356800 79356898 99 browser details YourSeq 93 368 466 3000 97.0% chr3 + 50703272 50703370 99 browser details YourSeq 93 368 472 3000 94.3% chr18 + 73136292 73136396 105 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% chr5 - 110263837 110266836 3000 browser details YourSeq 603 2352 2999 3000 96.9% chr6 + 31957652 31958300 649 browser details YourSeq 598 2354 3000 3000 96.6% chr16 - 43866466 43867112 647 browser details YourSeq 597 2343 2998 3000 96.0% chr15 + 11580640 11581309 670 browser details YourSeq 596 2354 2999 3000 96.0% chr9 + 77972796 77973438 643 browser details YourSeq 594 2358 2999 3000 96.3% chr2 - 136421939 136422579 641 browser details YourSeq 593 2358 2999 3000 96.7% chr6 - 52335414 52336055 642 browser details YourSeq 592 2353 2999 3000 95.8% chr7 - 57303384 57304029 646 browser details YourSeq 592 2356 2996 3000 96.1% chr3 + 7106711 7107347 637 browser details YourSeq 592 2358 2999 3000 96.1% chr19 + 54934365 54935005 641 browser details YourSeq 590 2352 2999 3000 95.5% chrX + 121899504 121900150 647 browser details YourSeq 589 2358 2999 3000 96.6% chr18 - 3641541 3642186 646 browser details YourSeq 588 2358 2999 3000 95.8% chr3 - 129058897 129059537 641 browser details YourSeq 588 2295 2999 3000 95.3% chr2 - 145426566 145427301 736 browser details YourSeq 588 2353 2999 3000 95.9% chr10 + 117426882 117427528 647 browser details YourSeq 588 2358 2999 3000 95.8% chr1 + 74863187 74863827 641 browser details YourSeq 588 2358 2994 3000 96.6% chr1 + 16629233 16629869 637 browser details YourSeq 587 2358 2999 3000 96.0% chrX - 17701010 17701659 650 browser details YourSeq 587 2353 2999 3000 96.0% chr6 - 117946604 117947257 654 browser details YourSeq 587 2358 2999 3000 96.1% chr12 - 30771659 30772304 646 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Pgam5 phosphoglycerate mutase family member 5 [ Mus musculus (house mouse) ] Gene ID: 72542, updated on 12-Aug-2019 Gene summary Official Symbol Pgam5 provided by MGI Official Full Name phosphoglycerate mutase family member 5 provided by MGI Primary source MGI:MGI:1919792 See related Ensembl:ENSMUSG00000029500 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 2610528A17Rik Expression Ubiquitous expression in placenta adult (RPKM 16.9), CNS E18 (RPKM 16.7) and 28 other tissues See more Orthologs human all Genomic context Location: 5; 5 F See Pgam5 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (110259135..110269899, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (110688154..110698918, complement) Chromosome 5 - NC_000071.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Pgam5 ENSMUSG00000029500 Description phosphoglycerate mutase family member 5 [Source:MGI Symbol;Acc:MGI:1919792] Gene Synonyms 2610528A17Rik Location Chromosome 5: 110,259,130-110,269,913 reverse strand. GRCm38:CM000998.2 About this gene This gene has 4 transcripts (splice variants), 197 orthologues, is a member of 1 Ensembl protein family and is associated with 36 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pgam5-202 ENSMUST00000112505.8 2098 288aa ENSMUSP00000108124.2 Protein coding CCDS51606 Q8BX10 TSL:1 GENCODE basic APPRIS ALT1 Pgam5-201 ENSMUST00000059229.15 2044 287aa ENSMUSP00000057760.8 Protein coding CCDS19523 Q8BX10 TSL:1 GENCODE basic APPRIS P3 Pgam5-204 ENSMUST00000196381.1 553 179aa ENSMUSP00000143477.1 Protein coding - A0A0G2JG95 CDS 3' incomplete TSL:5 Pgam5-203 ENSMUST00000136738.2 620 No protein - Retained intron - - TSL:3 30.78 kb Forward strand 110.25Mb 110.26Mb 110.27Mb Genes Ankle2-202 >protein coding Gm15788-202 >lncRNA (Comprehensive set... Ankle2-206 >protein coding Gm15788-201 >lncRNA Ankle2-201 >protein coding Ankle2-203 >lncRNA Contigs AC118260.7 > Genes (Comprehensive set... < Pgam5-202protein coding < Pxmp2-204nonsense mediated decay < Pgam5-201protein coding < Pxmp2-201protein coding < Pgam5-203retained intron < Pgam5-204protein coding Regulatory Build 110.25Mb 110.26Mb 110.27Mb Reverse strand 30.78 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000112505 < Pgam5-202protein coding Reverse strand 10.78 kb ENSMUSP00000108... Transmembrane heli... Low complexity (Seg) Superfamily Histidine phosphatase superfamily SMART Histidine phosphatase superfamily, clade-1 Pfam Histidine phosphatase superfamily, clade-1 PANTHER PTHR20935:SF0 PTHR20935 Gene3D Histidine phosphatase superfamily CDD Histidine phosphatase superfamily, clade-1 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 288 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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