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Mouse Phlda2 Knockout Project (CRISPR/Cas9)

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Mouse Phlda2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Phlda2 Knockout Project (CRISPR/Cas9) Objective: To create a Phlda2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Phlda2 gene (NCBI Reference Sequence: NM_009434 ; Ensembl: ENSMUSG00000010760 ) is located on Mouse chromosome 7. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000010904). Exon 1 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 disruptions in this gene have enlarged placentas but display little change in fetal weight. Exon 1 starts from about 0.23% of the coding region. Exon 1 covers 100.0% of the coding region. The size of effective KO region: ~430 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 Legends Exon of mouse Phlda2 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 start codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of stop codon is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. 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(19.75% 395) | C(27.5% 550) | T(23.1% 462) | G(29.65% 593) Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(22.25% 445) | C(24.35% 487) | T(25.9% 518) | G(27.5% 550) Note: The 2000 bp section downstream of stop codon 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% chr7 - 143502491 143504490 2000 browser details YourSeq 78 177 552 2000 87.7% chr18 + 76877933 76878342 410 browser details YourSeq 77 177 538 2000 76.2% chr5 - 125541723 125541865 143 browser details YourSeq 75 188 549 2000 74.8% chr11 - 88942981 88943252 272 browser details YourSeq 75 403 552 2000 93.3% chr12 + 11179941 11180412 472 browser details YourSeq 73 436 545 2000 92.1% chr9 - 78553487 78553596 110 browser details YourSeq 73 438 566 2000 84.3% chr7 + 67697515 67697636 122 browser details YourSeq 72 176 545 2000 76.0% chr9 - 37617422 37617588 167 browser details YourSeq 70 436 572 2000 84.3% chr19 - 29470109 29470240 132 browser details YourSeq 70 435 552 2000 92.8% chr17 - 4689392 4689510 119 browser details YourSeq 69 208 566 2000 73.5% chr1 + 179985557 179985744 188 browser details YourSeq 68 374 526 2000 93.6% chr6 - 28361098 28361470 373 browser details YourSeq 65 435 654 2000 88.0% chr15 - 93257473 93257689 217 browser details YourSeq 65 435 551 2000 93.4% chr1 - 157069030 157069147 118 browser details YourSeq 65 222 548 2000 71.5% chr12 + 23487782 23487927 146 browser details YourSeq 64 216 556 2000 91.2% chr19 + 44654403 44654746 344 browser details YourSeq 63 195 549 2000 73.0% chr11 - 100068131 100068319 189 browser details YourSeq 62 436 553 2000 91.5% chr5 - 112069310 112069426 117 browser details YourSeq 62 436 640 2000 90.8% chr3 + 70016107 70016449 343 browser details YourSeq 61 437 551 2000 93.0% chr14 - 49076801 49077328 528 Note: The 2000 bp section upstream of start codon 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% chr7 - 143500059 143502058 2000 browser details YourSeq 157 631 824 2000 89.5% chr8 + 13388070 13388253 184 browser details YourSeq 156 631 821 2000 88.5% chr11 - 79715892 79716073 182 browser details YourSeq 155 631 821 2000 90.6% chr4 + 136039751 136039936 186 browser details YourSeq 153 610 821 2000 89.6% chr4 - 126046338 126046533 196 browser details YourSeq 152 631 818 2000 89.3% chr1 - 180034035 180034216 182 browser details YourSeq 152 634 819 2000 91.8% chr17 + 66875619 66875805 187 browser details YourSeq 151 632 821 2000 91.1% chr2 - 90973671 90973859 189 browser details YourSeq 151 632 819 2000 88.7% chr11 + 100566190 100566366 177 browser details YourSeq 150 632 821 2000 92.2% chr9 - 50838686 50839082 397 browser details YourSeq 150 635 824 2000 91.7% chr4 + 41071389 41071587 199 browser details YourSeq 149 632 821 2000 88.8% chr9 - 103417410 103417594 185 browser details YourSeq 149 631 821 2000 90.3% chr1 + 71887357 72252865 365509 browser details YourSeq 148 632 820 2000 89.3% chr11 + 116426563 116426748 186 browser details YourSeq 147 632 821 2000 87.7% chr4 - 119157496 119157679 184 browser details YourSeq 147 631 821 2000 91.7% chr6 + 120594227 120594414 188 browser details YourSeq 145 625 821 2000 88.2% chr2 - 125252856 125253047 192 browser details YourSeq 145 632 821 2000 88.3% chr1 - 167318156 167318342 187 browser details YourSeq 145 632 821 2000 87.7% chr4 + 88813058 88813242 185 browser details YourSeq 144 636 821 2000 90.4% chr11 - 96791447 96791635 189 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Phlda2 pleckstrin homology like domain, family A, member 2 [ Mus musculus (house mouse) ] Gene ID: 22113, updated on 10-Oct-2019 Gene summary Official Symbol Phlda2 provided by MGI Official Full Name pleckstrin homology like domain, family A, member 2 provided by MGI Primary source MGI:MGI:1202307 See related Ensembl:ENSMUSG00000010760 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Ipl; Tssc3 Summary This gene is one of several genes in the imprinted gene domain on chromosome 7. Studies using knockout mice have Expression shown that the product of this gene regulates placental growth. Transcripts from this gene are most abundant in placenta and yolk sac, and are almost entirely transcribed from the maternal allele. [provided by RefSeq, Jul 2008] Orthologs Biased expression in placenta adult (RPKM 163.8), kidney adult (RPKM 10.9) and 1 other tissue See more human all Genomic context Location: 7 F5; 7 88.23 cM See Phlda2 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (143501545..143503145, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (150687453..150688429, complement) Chromosome 7 - NC_000073.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Phlda2 ENSMUSG00000010760 Description pleckstrin homology like domain, family A, member 2 [Source:MGI Symbol;Acc:MGI:1202307] Gene Synonyms Ipl, Tssc3 Location Chromosome 7: 143,501,545-143,503,150 reverse strand. GRCm38:CM001000.2 About this gene This gene has 3 transcripts (splice variants), 147 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Phlda2-201 ENSMUST00000010904.4 757 144aa ENSMUSP00000010904.3 Protein coding CCDS22042 O08969 TSL:1 GENCODE basic APPRIS P1 Phlda2-202 ENSMUST00000207425.1 376 105aa ENSMUSP00000146883.1 Protein coding - A0A140LIM7 CDS 3' incomplete TSL:3 Phlda2-203 ENSMUST00000207561.1 363 No protein - lncRNA - - TSL:3 21.61 kb Forward strand 143.495Mb 143.500Mb 143.505Mb 143.510Mb Genes Slc22a18-201 >protein coding (Comprehensive set... Slc22a18-205 >nonsense mediated decay Slc22a18-204 >nonsense mediated decay Slc22a18-203 >nonsense mediated decay Slc22a18-202 >protein coding Contigs < AC023248.20 Genes < Phlda2-201protein coding (Comprehensive set... < Phlda2-202protein coding < Phlda2-203lncRNA Regulatory Build 143.495Mb 143.500Mb 143.505Mb 143.510Mb Reverse strand 21.61 kb Regulation Legend CTCF Enhancer 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: ENSMUST00000010904 < Phlda2-201protein coding Reverse strand 997 bp ENSMUSP00000010... Superfamily SSF50729 SMART Pleckstrin homology domain Pfam PHLA2/3, PH domain PANTHER PTHR15478:SF8 PTHR15478 Gene3D PH-like domain superfamily CDD cd00821 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 20 40 60 80 100 120 144 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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