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Mouse Pla2g1b Conditional Knockout Project (CRISPR/Cas9)

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Mouse Pla2g1b Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pla2g1b Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pla2g1b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pla2g1b gene (NCBI Reference Sequence: NM_011107 ; Ensembl: ENSMUSG00000029522 ) is located on Mouse chromosome 5. 4 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 4 (Transcript: ENSMUST00000031495). 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 Pla2g1b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-87P14 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 display abnormalities in lipid absorption, increased insulin sensitivity and improved glucose tolerance. Exon 2 starts from about 7.99% 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: 4461 bp, and the size of intron 2 for 3'-loxP site insertion: 1051 bp. The size of effective cKO region: ~660 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 gRNA region 5' gRNA region 3' 1 2 3 4 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pla2g1b 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(7160bp) | A(28.17% 2017) | C(24.43% 1749) | T(24.92% 1784) | G(22.49% 1610) 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% chr5 + 115467533 115470532 3000 browser details YourSeq 334 1799 2180 3000 96.5% chr11 - 52390900 52391291 392 browser details YourSeq 317 1800 2171 3000 93.9% chr15 + 83188828 83189187 360 browser details YourSeq 316 1799 2180 3000 96.3% chr11 - 115735320 116169946 434627 browser details YourSeq 315 1796 2175 3000 94.5% chr5 - 146269916 146270289 374 browser details YourSeq 315 1799 2167 3000 96.5% chr5 + 136726511 136727043 533 browser details YourSeq 304 1799 2180 3000 96.1% chr15 - 8510631 8511181 551 browser details YourSeq 301 1799 2169 3000 90.6% chr1 - 58452390 58452756 367 browser details YourSeq 298 1795 2178 3000 91.8% chr17 - 78776534 78776901 368 browser details YourSeq 295 1798 2178 3000 93.3% chr4 - 134875098 134875477 380 browser details YourSeq 290 1798 2160 3000 95.1% chr4 - 140683015 140683607 593 browser details YourSeq 286 1799 2170 3000 96.2% chr16 - 14322518 14323131 614 browser details YourSeq 286 1799 2165 3000 93.7% chr7 + 12999956 13000319 364 browser details YourSeq 281 1798 2159 3000 95.5% chr7 + 3187461 3188040 580 browser details YourSeq 279 1799 2151 3000 95.5% chr1 - 164024582 164025081 500 browser details YourSeq 271 1799 2157 3000 95.1% chr4 + 132222276 132222794 519 browser details YourSeq 270 1799 2161 3000 93.5% chr17 - 5276320 5276666 347 browser details YourSeq 270 1799 2153 3000 94.5% chr7 + 45532941 45533342 402 browser details YourSeq 270 1799 2171 3000 94.8% chr17 + 32261386 32261762 377 browser details YourSeq 268 1799 2168 3000 93.3% chrX - 52150095 52150541 447 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 + 115471193 115474192 3000 browser details YourSeq 154 2223 2647 3000 83.6% chr12 - 69204880 69205208 329 browser details YourSeq 148 269 572 3000 88.2% chrX + 60222611 60223016 406 browser details YourSeq 146 270 574 3000 88.3% chr1 + 194191435 194191743 309 browser details YourSeq 142 401 573 3000 92.9% chr4 + 56955041 56955491 451 browser details YourSeq 134 2152 2634 3000 82.2% chr15 + 12229480 12229734 255 browser details YourSeq 128 401 593 3000 90.6% chr4 - 135980070 135980333 264 browser details YourSeq 128 2155 2647 3000 82.3% chr2 + 180079793 180080197 405 browser details YourSeq 127 2152 2346 3000 86.0% chr4 - 156018234 156018375 142 browser details YourSeq 127 2155 2351 3000 86.0% chr13 - 58162093 58162235 143 browser details YourSeq 125 2143 2341 3000 84.8% chr3 - 51196273 51196417 145 browser details YourSeq 125 2142 2341 3000 85.4% chr17 - 72594627 72594780 154 browser details YourSeq 125 2152 2346 3000 85.9% chr10 + 21088745 21088886 142 browser details YourSeq 124 1584 2166 3000 78.5% chr11 - 86654901 86655117 217 browser details YourSeq 124 2153 2346 3000 85.2% chr3 + 121432677 121432817 141 browser details YourSeq 124 2152 2348 3000 86.2% chr16 + 18688394 18688536 143 browser details YourSeq 122 2152 2341 3000 86.1% chr3 - 121431567 121431703 137 browser details YourSeq 122 2152 2347 3000 84.6% chrX + 94034538 94034680 143 browser details YourSeq 120 2152 2341 3000 85.3% chr7 - 27100426 27100562 137 browser details YourSeq 120 2152 2347 3000 85.9% chr13 - 91216373 91216515 143 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: Pla2g1b phospholipase A2, group IB, pancreas [ Mus musculus (house mouse) ] Gene ID: 18778, updated on 24-Oct-2019 Gene summary Official Symbol Pla2g1b provided by MGI Official Full Name phospholipase A2, group IB, pancreas provided by MGI Primary source MGI:MGI:101842 See related Ensembl:ENSMUSG00000029522 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 Pla2a; sPLA2IB Expression Restricted expression toward stomach adult (RPKM 11970.9) See more Orthologs human all Genomic context Location: 5; 5 F See Pla2g1b in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (115466142..115474722) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (115916275..115924726) Chromosome 5 - NC_000071.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Pla2g1b ENSMUSG00000029522 Description phospholipase A2, group IB, pancreas [Source:MGI Symbol;Acc:MGI:101842] Gene Synonyms Pla2a, sPLA2IB Location Chromosome 5: 115,466,262-115,474,722 forward strand. GRCm38:CM000998.2 About this gene This gene has 5 transcripts (splice variants), 263 orthologues, 8 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pla2g1b-201 ENSMUST00000031495.10 557 146aa ENSMUSP00000031495.4 Protein coding CCDS19592 Q9Z0Y2 TSL:1 GENCODE basic APPRIS P1 Pla2g1b-204 ENSMUST00000145785.7 419 77aa ENSMUSP00000138683.1 Protein coding - S4R2K6 TSL:5 GENCODE basic Pla2g1b-203 ENSMUST00000125568.1 387 124aa ENSMUSP00000120743.1 Protein coding - D3YWH2 CDS 3' incomplete TSL:2 Pla2g1b-202 ENSMUST00000112071.7 361 82aa ENSMUSP00000107702.1 Protein coding - D3Z1N8 TSL:1 GENCODE basic Pla2g1b-205 ENSMUST00000202822.1 371 No protein - lncRNA - - TSL:1 Page 6 of 8 https://www.alphaknockout.com 28.46 kb Forward strand 115.46Mb 115.47Mb 115.48Mb Genes (Comprehensive set... Pla2g1b-204 >protein coding Pla2g1b-201 >protein coding Pla2g1b-205 >lncRNA Pla2g1b-203 >protein coding Pla2g1b-202 >protein coding Contigs < AC117735.8 AC159539.6 > Genes < Sirt4-201protein coding (Comprehensive set... < Sirt4-202protein coding < Sirt4-206retained intron < Sirt4-204retained intron < Sirt4-205lncRNA < Sirt4-203retained intron Regulatory Build 115.46Mb 115.47Mb 115.48Mb Reverse strand 28.46 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: ENSMUST00000031495 8.46 kb Forward strand Pla2g1b-201 >protein coding ENSMUSP00000031... Low complexity (Seg) Cleavage site (Sign... Superfamily Phospholipase A2 domain superfamily SMART Phospholipase A2 domain Prints Phospholipase A2 Pfam Phospholipase A2 domain PROSITE patterns Phospholipase A2, histidine active site Phospholipase A2, aspartic acid active site PANTHER PTHR11716:SF84 Phospholipase A2 Gene3D Phospholipase A2 domain superfamily CDD Phospholipase A2 domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 146 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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