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Mouse Pcgf5 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Pcgf5 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pcgf5 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pcgf5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pcgf5 gene (NCBI Reference Sequence: NM_029508 ; Ensembl: ENSMUSG00000024805 ) is located on Mouse chromosome 19. 9 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000071267). 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 Pcgf5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-148L3 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: Bone marrow cells from mice homozygous for a conditional allele exhibit normal hematopoietic and progenitor cell function. Exon 3 starts from about 15.96% 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: 22333 bp, and the size of intron 3 for 3'-loxP site insertion: 2488 bp. The size of effective cKO region: ~597 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 3 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pcgf5 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. 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(7097bp) | A(29.73% 2110) | C(18.44% 1309) | T(28.63% 2032) | G(23.19% 1646) 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% chr19 + 36431321 36434320 3000 browser details YourSeq 70 760 947 3000 86.5% chr9 + 122498224 122498443 220 browser details YourSeq 62 661 801 3000 83.8% chr3 + 122514240 122514374 135 browser details YourSeq 61 674 831 3000 69.5% chr14 + 33852707 33852864 158 browser details YourSeq 54 685 942 3000 93.6% chr2 - 76837684 76838271 588 browser details YourSeq 52 685 792 3000 93.4% chr11 + 54923873 54923980 108 browser details YourSeq 51 685 794 3000 96.5% chr15 - 76849135 76849688 554 browser details YourSeq 50 680 782 3000 93.2% chr14 - 47438080 47438182 103 browser details YourSeq 46 689 801 3000 81.9% chr7 + 18917371 18917482 112 browser details YourSeq 46 752 820 3000 84.1% chr5 + 147817766 147818306 541 browser details YourSeq 46 686 796 3000 91.1% chr1 + 37348048 37348158 111 browser details YourSeq 45 685 782 3000 94.2% chr8 - 24589104 24589201 98 browser details YourSeq 45 657 782 3000 79.3% chr7 - 99615874 99615989 116 browser details YourSeq 45 685 778 3000 96.0% chr10 - 34178353 34178446 94 browser details YourSeq 45 659 785 3000 80.4% chr14 + 74859949 74860064 116 browser details YourSeq 44 758 823 3000 83.4% chr11 - 87723032 87723097 66 browser details YourSeq 44 685 777 3000 95.9% chr4 + 135447463 135447555 93 browser details YourSeq 43 780 830 3000 88.0% chr2 - 122157496 122157545 50 browser details YourSeq 43 685 782 3000 93.9% chr2 - 66595690 66595787 98 browser details YourSeq 43 682 778 3000 89.8% chr17 - 17051677 17051772 96 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% chr19 + 36434918 36437917 3000 browser details YourSeq 132 1909 2058 3000 94.0% chr3 - 52290560 52290709 150 browser details YourSeq 124 1920 2058 3000 95.6% chr16 - 18366134 18366272 139 browser details YourSeq 123 1924 2062 3000 95.6% chr7 + 38253119 38253277 159 browser details YourSeq 120 1920 2057 3000 91.2% chr17 - 35564433 35564567 135 browser details YourSeq 119 1924 2058 3000 94.1% chr12 + 76391475 76391609 135 browser details YourSeq 117 1924 2061 3000 92.8% chr1 - 152915390 152915533 144 browser details YourSeq 117 1924 2058 3000 91.0% chr11 + 97155911 97156042 132 browser details YourSeq 116 1924 2058 3000 92.5% chr10 - 18220344 18220477 134 browser details YourSeq 115 1924 2054 3000 91.5% chr3 + 20101451 20101578 128 browser details YourSeq 113 1926 2058 3000 90.0% chr19 - 45382109 45382238 130 browser details YourSeq 113 1924 2058 3000 89.4% chrX + 139706442 139706573 132 browser details YourSeq 112 1929 2061 3000 93.1% chr3 - 106151257 106151393 137 browser details YourSeq 112 1930 2058 3000 93.6% chr17 + 24269460 24269587 128 browser details YourSeq 111 1926 2058 3000 94.5% chr8 - 78623628 78623764 137 browser details YourSeq 111 1929 2057 3000 90.5% chr1 - 80386966 80387091 126 browser details YourSeq 111 1930 2060 3000 92.4% chr1 - 13620914 13621044 131 browser details YourSeq 111 1929 2055 3000 91.0% chr13 + 40818999 40819120 122 browser details YourSeq 110 1929 2052 3000 90.9% chr11 - 17602854 17602973 120 browser details YourSeq 110 1930 2058 3000 91.2% chrX + 155352875 155353000 126 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Pcgf5 polycomb group ring finger 5 [ Mus musculus (house mouse) ] Gene ID: 76073, updated on 24-Oct-2019 Gene summary Official Symbol Pcgf5 provided by MGI Official Full Name polycomb group ring finger 5 provided by MGI Primary source MGI:MGI:1923505 See related Ensembl:ENSMUSG00000024805 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 AI324127; 0610009F02Rik; 1110054A01Rik; 5830406C17Rik; 5830443C21Rik; 9530023M17Rik Expression Ubiquitous expression in placenta adult (RPKM 8.9), heart adult (RPKM 3.9) and 26 other tissues See more Orthologs human all Genomic context Location: 19; 19 C2 See Pcgf5 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (36348329..36460970) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (36453557..36530694) Chromosome 19 - NC_000085.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 11 transcripts Gene: Pcgf5 ENSMUSG00000024805 Description polycomb group ring finger 5 [Source:MGI Symbol;Acc:MGI:1923505] Gene Synonyms 0610009F02Rik, 1110054A01Rik, 5830406C17Rik, 5830443C21Rik, 9530023M17Rik Location Chromosome 19: 36,348,309-36,460,970 forward strand. GRCm38:CM001012.2 About this gene This gene has 11 transcripts (splice variants), 205 orthologues, 7 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pcgf5-211 ENSMUST00000225920.1 6443 236aa ENSMUSP00000153206.1 Protein coding CCDS29772 Q3UK78 GENCODE basic APPRIS P2 Pcgf5-201 ENSMUST00000062389.5 1566 236aa ENSMUSP00000058730.5 Protein coding CCDS29772 Q3UK78 TSL:5 GENCODE basic APPRIS P2 Pcgf5-202 ENSMUST00000071267.13 1508 236aa ENSMUSP00000071245.6 Protein coding CCDS29772 Q3UK78 TSL:1 GENCODE basic APPRIS P2 Pcgf5-210 ENSMUST00000225411.1 1626 256aa ENSMUSP00000153464.1 Protein coding - Q3UK78 GENCODE basic APPRIS ALT1 Pcgf5-203 ENSMUST00000224679.1 1269 256aa ENSMUSP00000153681.1 Protein coding - Q3UK78 GENCODE basic APPRIS ALT1 Pcgf5-204 ENSMUST00000224772.1 1266 255aa ENSMUSP00000153066.1 Protein coding - B7ZP24 GENCODE basic APPRIS ALT1 Pcgf5-207 ENSMUST00000224971.1 599 125aa ENSMUSP00000153342.1 Protein coding - A0A286YDQ6 CDS 3' incomplete Pcgf5-206 ENSMUST00000224859.1 430 132aa ENSMUSP00000153211.1 Protein coding - A0A286YD06 CDS 5' incomplete Pcgf5-209 ENSMUST00000225185.1 3941 No protein - Retained intron - - - Pcgf5-208 ENSMUST00000225050.1 506 No protein - Retained intron - - - Pcgf5-205 ENSMUST00000224805.1 1824 No protein - lncRNA - - - Page 6 of 8 https://www.alphaknockout.com 132.66 kb Forward strand 36.34Mb 36.36Mb 36.38Mb 36.40Mb 36.42Mb 36.44Mb 36.46Mb Genes (Comprehensive set... Pcgf5-211 >protein coding Pcgf5-205 >lncRNA Pcgf5-202 >protein coding Gm47773-201 >processed pseudogene Pcgf5-207 >protein coding Pcgf5-203 >protein coding Pcgf5-204 >protein coding Gm9042-201 >processed pseudogene Pcgf5-208 >retained intron Pcgf5-201 >protein coding Pcgf5-210 >protein coding Pcgf5-206 >protein coding Pcgf5-209 >retained intron Contigs < AC117769.9 Genes < Gm47735-201lncRNA < Gm23170-201snRNA
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