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Mouse Pdlim1 Knockout Project (CRISPR/Cas9)

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Mouse Pdlim1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pdlim1 Knockout Project (CRISPR/Cas9) Objective: To create a Pdlim1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pdlim1 gene (NCBI Reference Sequence: NM_016861 ; Ensembl: ENSMUSG00000055044 ) is located on Mouse chromosome 19. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000068439). Exon 2~4 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a gene trap allele exhibit enhanced platelet response to GPVI agonists and thrombosis. Exon 2 starts from about 9.89% of the coding region. Exon 2~4 covers 43.93% of the coding region. The size of effective KO region: ~8656 bp. The KO 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 3 4 7 Legends Exon of mouse Pdlim1 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 4 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(25.35% 507) | C(22.05% 441) | T(28.5% 570) | G(24.1% 482) Note: The 2000 bp section upstream of Exon 2 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.05% 481) | C(21.25% 425) | T(27.4% 548) | G(27.3% 546) Note: The 2000 bp section downstream of Exon 4 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr19 - 40252062 40254061 2000 browser details YourSeq 229 1392 1792 2000 88.1% chr8 - 85707086 85707484 399 browser details YourSeq 224 1400 1767 2000 89.6% chr2 + 118953182 118953547 366 browser details YourSeq 217 1395 1766 2000 85.8% chr7 - 34488597 34488934 338 browser details YourSeq 216 1391 1758 2000 89.2% chr2 - 126456218 126456585 368 browser details YourSeq 215 1391 1767 2000 87.9% chr8 + 41209097 41209472 376 browser details YourSeq 215 1433 1761 2000 86.6% chr7 + 79061802 79062098 297 browser details YourSeq 212 1401 1761 2000 85.0% chr3 - 9486466 9486820 355 browser details YourSeq 212 1390 1766 2000 88.9% chr11 - 54078354 54078749 396 browser details YourSeq 212 1392 1766 2000 89.3% chr4 + 46217186 46217566 381 browser details YourSeq 211 1392 1761 2000 87.4% chr13 + 91267135 91267491 357 browser details YourSeq 209 1392 1766 2000 88.4% chr12 - 84816644 84817013 370 browser details YourSeq 209 1392 1756 2000 86.3% chr1 - 183237363 183237696 334 browser details YourSeq 209 1392 1761 2000 85.5% chr11 + 104862662 104863027 366 browser details YourSeq 209 1391 1726 2000 90.8% chr1 + 91140449 91140803 355 browser details YourSeq 207 1391 1778 2000 85.1% chr16 - 55872070 55872405 336 browser details YourSeq 205 1392 1773 2000 85.7% chr5 + 65838500 65838840 341 browser details YourSeq 203 1402 1756 2000 87.7% chr16 + 37803679 37804030 352 browser details YourSeq 202 1391 1763 2000 92.2% chr8 - 87320578 87320960 383 browser details YourSeq 202 1392 1755 2000 83.3% chr4 - 45328645 45328967 323 Note: The 2000 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 2000 1 2000 2000 100.0% chr19 - 40241406 40243405 2000 browser details YourSeq 37 362 437 2000 95.3% chr7 + 99005292 99005368 77 browser details YourSeq 29 1398 1434 2000 96.9% chr4 + 51484126 51484166 41 browser details YourSeq 27 1402 1434 2000 78.6% chr18 + 7091409 7091436 28 browser details YourSeq 23 397 443 2000 74.5% chr11 + 5389351 5389397 47 browser details YourSeq 22 27 48 2000 100.0% chr7 - 139351060 139351081 22 browser details YourSeq 22 282 306 2000 96.0% chr13 + 118189296 118189322 27 browser details YourSeq 21 1414 1434 2000 100.0% chr12 - 17660121 17660141 21 browser details YourSeq 21 1414 1434 2000 100.0% chr10 + 24301186 24301206 21 Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Pdlim1 PDZ and LIM domain 1 (elfin) [ Mus musculus (house mouse) ] Gene ID: 54132, updated on 10-Oct-2019 Gene summary Official Symbol Pdlim1 provided by MGI Official Full Name PDZ and LIM domain 1 (elfin) provided by MGI Primary source MGI:MGI:1860611 See related Ensembl:ENSMUSG00000055044 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 CLP36; Clim1; mClim1 Expression Broad expression in large intestine adult (RPKM 123.7), placenta adult (RPKM 92.4) and 19 other tissues See more Orthologs human all Genomic context Location: 19; 19 C3 See Pdlim1 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (40222239..40271616, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (40296729..40346106, complement) Chromosome 19 - NC_000085.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Pdlim1 ENSMUSG00000055044 Description PDZ and LIM domain 1 (elfin) [Source:MGI Symbol;Acc:MGI:1860611] Gene Synonyms CLP36, mClim1 Location Chromosome 19: 40,221,173-40,271,842 reverse strand. GRCm38:CM001012.2 About this gene This gene has 5 transcripts (splice variants), 219 orthologues, 8 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pdlim1-201 ENSMUST00000068439.12 2571 327aa ENSMUSP00000064545.5 Protein coding CCDS37977 O70400 TSL:1 GENCODE basic APPRIS P1 Pdlim1-202 ENSMUST00000182432.1 692 198aa ENSMUSP00000138383.1 Protein coding - S4R1V0 CDS 3' incomplete TSL:3 Pdlim1-204 ENSMUST00000182636.1 2835 No protein - Retained intron - - TSL:1 Pdlim1-203 ENSMUST00000182540.1 715 No protein - Retained intron - - TSL:2 Pdlim1-205 ENSMUST00000182813.1 666 No protein - Retained intron - - TSL:2 70.67 kb Forward strand Genes Gm16470-201 >processed pseudogene (Comprehensive set... Contigs AC170187.2 > Genes (Comprehensive set... < Pdlim1-201protein coding < Pdlim1-203retained intron < Pdlim1-204retained intron < Pdlim1-205retained intron < Pdlim1-202protein coding Regulatory Build Reverse strand 70.67 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000068439 < Pdlim1-201protein coding Reverse strand 50.44 kb ENSMUSP00000064... Superfamily PDZ superfamily SSF57716 SMART PDZ domain Zasp-like motif Zinc finger, LIM-type Pfam PDZ domain Domain of unknown function DUF4749 Zinc finger, LIM-type PROSITE profiles PDZ domain Zinc finger, LIM-type PROSITE patterns Zinc finger, LIM-type PANTHER PTHR24214 PDZ and LIM domain protein 1 Gene3D 2.30.42.10 2.10.110.10 CDD cd00992 cd09448 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 327 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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