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Mouse Mgst3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Mgst3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Mgst3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Mgst3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mgst3 gene (NCBI Reference Sequence: NM_025569 ; Ensembl: ENSMUSG00000026688 ) is located on Mouse chromosome 1. 6 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000028005). 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 Mgst3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-279M3 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: 15335 bp, and the size of intron 2 for 3'-loxP site insertion: 957 bp. The size of effective cKO region: ~617 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 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Mgst3 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(7117bp) | A(25.57% 1820) | C(22.8% 1623) | T(27.17% 1934) | G(24.45% 1740) 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 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% chr1 - 167378659 167381658 3000 browser details YourSeq 194 1922 2142 3000 93.7% chr1 - 167380870 167381074 205 browser details YourSeq 194 585 789 3000 97.6% chr1 - 167379517 167379737 221 browser details YourSeq 92 387 1076 3000 82.9% chr11 + 70034610 70131762 97153 browser details YourSeq 70 2368 2835 3000 77.4% chr1 + 56851978 56852406 429 browser details YourSeq 62 389 488 3000 88.8% chr1 - 13239574 13239684 111 browser details YourSeq 61 2360 2833 3000 93.1% chr16 - 93998393 93998933 541 browser details YourSeq 58 1016 1083 3000 94.2% chr1 - 133819018 133870668 51651 browser details YourSeq 54 1010 1082 3000 84.6% chr11 + 5436803 5436874 72 browser details YourSeq 53 1000 1075 3000 85.4% chr4 - 57823643 57823719 77 browser details YourSeq 52 388 460 3000 86.2% chr5 - 43804091 43804172 82 browser details YourSeq 51 396 487 3000 93.2% chr7 - 113032803 113032902 100 browser details YourSeq 51 391 483 3000 91.9% chr12 - 55330624 55330716 93 browser details YourSeq 47 1000 1080 3000 81.4% chr17 - 26474864 26474944 81 browser details YourSeq 46 380 444 3000 85.0% chr6 + 149453996 149454059 64 browser details YourSeq 45 1000 1082 3000 92.6% chr15 + 31407097 31407181 85 browser details YourSeq 44 396 488 3000 86.7% chr6 - 51263175 51263276 102 browser details YourSeq 44 1007 1086 3000 96.0% chr8 + 116330214 116330294 81 browser details YourSeq 43 396 486 3000 89.1% chr1 + 58398195 58398453 259 browser details YourSeq 42 1017 1083 3000 82.1% chr12 - 25362274 25362341 68 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% chr1 - 167375042 167378041 3000 browser details YourSeq 112 2230 2483 3000 82.4% chr5 + 55027661 55027899 239 browser details YourSeq 112 2332 2483 3000 89.4% chr10 + 41446904 41447223 320 browser details YourSeq 107 2302 2483 3000 79.5% chr7 + 44235124 44235271 148 browser details YourSeq 106 2299 2487 3000 84.2% chr7 + 126094710 126094868 159 browser details YourSeq 103 2332 2487 3000 82.6% chrX + 82333607 82333748 142 browser details YourSeq 103 2317 2487 3000 90.8% chr12 + 100181500 100182109 610 browser details YourSeq 101 2295 2483 3000 81.9% chr2 - 127294632 127294796 165 browser details YourSeq 101 1177 1312 3000 87.5% chr1 - 40932606 40932737 132 browser details YourSeq 100 2336 2487 3000 82.2% chr6 + 87174481 87174618 138 browser details YourSeq 98 2299 2483 3000 80.5% chr15 + 26002092 26002238 147 browser details YourSeq 97 2302 2483 3000 81.6% chr4 - 143269736 143269899 164 browser details YourSeq 95 2331 2479 3000 83.2% chr16 + 20277795 20277929 135 browser details YourSeq 95 2333 2484 3000 79.9% chr12 + 110495738 110495869 132 browser details YourSeq 95 2333 2487 3000 81.6% chr12 + 75901663 75901803 141 browser details YourSeq 94 2331 2483 3000 83.2% chr18 - 19025408 19025545 138 browser details YourSeq 93 2332 2493 3000 82.4% chr19 - 10862850 10863001 152 browser details YourSeq 93 2331 2487 3000 81.2% chr10 - 62384374 62384516 143 browser details YourSeq 93 2331 2487 3000 83.5% chr16 + 17811326 17811466 141 browser details YourSeq 92 2337 2487 3000 81.7% chr11 - 72788242 72788378 137 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: Mgst3 microsomal glutathione S-transferase 3 [ Mus musculus (house mouse) ] Gene ID: 66447, updated on 12-Aug-2019 Gene summary Official Symbol Mgst3 provided by MGI Official Full Name microsomal glutathione S-transferase 3 provided by MGI Primary source MGI:MGI:1913697 See related Ensembl:ENSMUSG00000026688 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 GST-III; AA516734; 2010012L10Rik; 2010306B17Rik; 2700004G04Rik Expression Broad expression in stomach adult (RPKM 1980.6), duodenum adult (RPKM 1260.0) and 18 other tissues See more Orthologs human all Genomic context Location: 1; 1 H2.3 See Mgst3 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (167371966..167393841, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (169302515..169323928, complement) Chromosome 1 - NC_000067.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Mgst3 ENSMUSG00000026688 Description microsomal glutathione S-transferase 3 [Source:MGI Symbol;Acc:MGI:1913697] Gene Synonyms 2010012L10Rik, 2010306B17Rik, 2700004G04Rik, GST-III Location Chromosome 1: 167,371,966-167,393,841 reverse strand. GRCm38:CM000994.2 About this gene This gene has 1 transcript (splice variant), 200 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mgst3-201 ENSMUST00000028005.2 1097 153aa ENSMUSP00000028005.2 Protein coding CCDS15457 Q9CPU4 TSL:1 GENCODE basic APPRIS P1 41.88 kb Forward strand 167.37Mb 167.38Mb 167.39Mb 167.40Mb Genes Aldh9a1-201 >protein coding (Comprehensive set... Aldh9a1-202 >retained intron Contigs AC113970.8 > Genes < Mgst3-201protein coding (Comprehensive set... Regulatory Build 167.37Mb 167.38Mb 167.39Mb 167.40Mb Reverse strand 41.88 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000028005 < Mgst3-201protein coding Reverse strand 21.88 kb ENSMUSP00000028... Transmembrane heli... Superfamily Membrane associated eicosanoid/glutathione metabolism-like domain superfamily Pfam Membrane-associated, eicosanoid/glutathione metabolism (MAPEG) protein PANTHER PTHR10250 PTHR10250:SF21 Gene3D Membrane associated eicosanoid/glutathione metabolism-like domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 153 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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