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Mouse Pik3cb Conditional Knockout Project (CRISPR/Cas9)

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Mouse Pik3cb Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pik3cb Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pik3cb conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pik3cb gene (NCBI Reference Sequence: NM_029094 ; Ensembl: ENSMUSG00000032462 ) is located on Mouse chromosome 9. 24 exons are identified, with the ATG start codon in exon 3 and the TAG stop codon in exon 24 (Transcript: ENSMUST00000035037). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pik3cb gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-349B12 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 a knock-out allele exhibit 30% fetal lethality, decreased size at birth and postnatally, abnormal glucose homeostasis, and dyslipidemia. Mice homozygous for a different knock-out allele die prior to E8.5. Exon 3 starts from about 100% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 16566 bp, and the size of intron 3 for 3'-loxP site insertion: 4130 bp. The size of effective cKO region: ~653 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 3 24 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pik3cb 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(7153bp) | A(25.12% 1797) | C(21.08% 1508) | T(31.83% 2277) | G(21.96% 1571) 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% chr9 - 99105934 99108933 3000 browser details YourSeq 296 1412 2093 3000 87.1% chr18 + 4607298 4607834 537 browser details YourSeq 291 1602 2318 3000 85.9% chr17 - 26591301 26591920 620 browser details YourSeq 277 1883 2284 3000 91.6% chr18 - 4477762 4478432 671 browser details YourSeq 267 1866 2335 3000 89.0% chr13 - 55334771 55335320 550 browser details YourSeq 259 1586 2091 3000 86.7% chrX - 72879783 72880329 547 browser details YourSeq 247 1588 2091 3000 86.7% chr11 + 25194813 25195337 525 browser details YourSeq 246 1597 2091 3000 88.0% chr19 + 58142581 58143096 516 browser details YourSeq 245 1585 2093 3000 91.0% chr9 - 73148893 73149430 538 browser details YourSeq 238 1582 2138 3000 88.9% chr1 - 33844142 33844850 709 browser details YourSeq 237 1590 2057 3000 84.8% chr7 - 140192031 140192453 423 browser details YourSeq 237 1582 2094 3000 86.8% chr15 - 66985741 66986477 737 browser details YourSeq 234 1509 2082 3000 90.4% chr14 + 48269527 48270222 696 browser details YourSeq 231 1589 2091 3000 88.4% chr15 + 80815649 80816175 527 browser details YourSeq 230 2223 2786 3000 90.5% chr8 - 86836974 86837285 312 browser details YourSeq 230 1590 2087 3000 86.0% chr4 - 58933309 58933796 488 browser details YourSeq 230 1595 2091 3000 88.6% chr2 - 125237405 125237926 522 browser details YourSeq 229 1582 2091 3000 89.6% chr7 + 128789334 128789942 609 browser details YourSeq 228 1580 2084 3000 89.0% chr3 - 87639490 88053198 413709 browser details YourSeq 227 1589 2380 3000 90.1% chr2 - 146055794 146056797 1004 Note: The 3000 bp section upstream of Exon 3 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% chr9 - 99102281 99105280 3000 browser details YourSeq 144 1 159 3000 97.4% chr2 - 154659957 154660135 179 browser details YourSeq 140 13 159 3000 98.0% chr15 + 8717681 8717829 149 browser details YourSeq 138 13 159 3000 97.3% chrX - 153272302 153272452 151 browser details YourSeq 137 13 159 3000 97.3% chr5 - 121202255 121202403 149 browser details YourSeq 137 13 159 3000 98.0% chr15 + 10573271 10573445 175 browser details YourSeq 135 1 159 3000 91.3% chr17 + 31529257 31529409 153 browser details YourSeq 134 4 159 3000 95.4% chr2 - 152855781 152856325 545 browser details YourSeq 133 646 2937 3000 88.1% chr1 - 55008224 55253334 245111 browser details YourSeq 132 13 153 3000 97.2% chr4 + 32799605 32799747 143 browser details YourSeq 131 13 159 3000 95.3% chr14 - 62801176 62801339 164 browser details YourSeq 131 13 160 3000 94.6% chr13 - 67676812 67676961 150 browser details YourSeq 131 13 157 3000 95.9% chr6 + 38632614 38632760 147 browser details YourSeq 130 13 159 3000 95.2% chr8 - 80968757 80968903 147 browser details YourSeq 130 13 156 3000 95.8% chr5 - 67278987 67279131 145 browser details YourSeq 130 13 158 3000 96.5% chr9 + 56979356 56979503 148 browser details YourSeq 130 13 159 3000 94.6% chr9 + 21119019 21119167 149 browser details YourSeq 130 1 148 3000 92.3% chr19 + 46243593 46243735 143 browser details YourSeq 129 7 141 3000 98.6% chr5 - 80349247 80349490 244 browser details YourSeq 128 13 156 3000 94.3% chr2 - 25244849 25244991 143 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Pik3cb phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta [ Mus musculus (house mouse) ] Gene ID: 74769, updated on 10-Oct-2019 Gene summary Official Symbol Pik3cb provided by MGI Official Full Name phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta provided by MGI Primary source MGI:MGI:1922019 See related Ensembl:ENSMUSG00000032462 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 AI447572; p110beta; 1110001J02Rik Expression Ubiquitous expression in placenta adult (RPKM 11.2), subcutaneous fat pad adult (RPKM 5.0) and 28 other tissues See Orthologs more human all Genomic context Location: 9; 9 E3.3 See Pik3cb in Genome Data Viewer Exon count: 25 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (99038402..99140235, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (98938821..99040630, complement) Chromosome 9 - NC_000075.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Pik3cb ENSMUSG00000032462 Description phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta [Source:MGI Symbol;Acc:MGI:1922019] Gene Synonyms 1110001J02Rik, p110beta Location Chromosome 9: 99,036,654-99,140,621 reverse strand. GRCm38:CM001002.2 About this gene This gene has 3 transcripts (splice variants), 210 orthologues, 9 paralogues, 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 Pik3cb- ENSMUST00000035037.13 6478 1064aa ENSMUSP00000035037.7 Protein coding CCDS23432 Q8BTI9 TSL:1 201 GENCODE basic APPRIS P1 Pik3cb- ENSMUST00000124723.1 631 68aa ENSMUSP00000121466.1 Protein coding - D3Z2Z7 CDS 3' 202 incomplete TSL:5 Pik3cb- ENSMUST00000136965.7 5045 530aa ENSMUSP00000138346.1 Nonsense mediated - S4R1S1 TSL:1 203 decay 123.97 kb Forward strand 99.04Mb 99.06Mb 99.08Mb 99.10Mb 99.12Mb 99.14Mb Contigs < AC120148.13 AC122930.4 > Genes < Gm1123-201protein coding < Pik3cb-202protein coding (Comprehensive set... < Pik3cb-201protein coding < Pik3cb-203nonsense mediated decay Regulatory Build 99.04Mb 99.06Mb 99.08Mb 99.10Mb 99.12Mb 99.14Mb Reverse strand 123.97 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000035037 < Pik3cb-201protein coding Reverse strand 103.43 kb ENSMUSP00000035... PDB-ENSP mappings Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF49562 Armadillo-type fold Protein kinase-like domain superfamily Ubiquitin-like domain superfamily SMART Phosphatidylinositol 3-kinase adaptor-binding (PI3K ABD) domain Phosphatidylinositol 3-/4-kinase, catalytic domain Phosphoinositide 3-kinase, accessory (PIK) domain Phosphatidylinositol 3-kinase, C2 domain Phosphatidylinositol 3-kinase Ras-binding (PI3K
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