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Mouse Vav3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Vav3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Vav3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Vav3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Vav3 gene (NCBI Reference Sequence: NM_020505 ; Ensembl: ENSMUSG00000033721 ) is located on Mouse chromosome 3. 27 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 27 (Transcript: ENSMUST00000046864). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Vav3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-79G21 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: Homozygous mutation of this gene results in tachycardia, systemic arterial hypertension, cardiovascular remodeling, hyperactivity of sympathetic neurons and thus high catecholamine levels, and increased levels of kidney- related hormones. Exon 3 starts from about 12.67% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 70670 bp, and the size of intron 4 for 3'-loxP site insertion: 4424 bp. The size of effective cKO region: ~2676 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 5' gRNA region gRNA region 3' 1 3 4 27 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Vav3 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(9176bp) | A(27.43% 2517) | C(20.3% 1863) | T(31.87% 2924) | G(20.4% 1872) 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% chr3 + 109491529 109494528 3000 browser details YourSeq 715 2057 2864 3000 94.6% chr5 - 67174701 67175529 829 browser details YourSeq 713 2056 2864 3000 95.1% chr18 - 22307972 22308803 832 browser details YourSeq 709 2054 2864 3000 94.1% chr19 - 14739188 14740014 827 browser details YourSeq 708 2057 2864 3000 94.4% chr18 - 32952101 32952931 831 browser details YourSeq 707 2054 2882 3000 93.6% chr18 - 22772051 22772909 859 browser details YourSeq 705 2058 2864 3000 94.3% chr8 + 28306041 28306881 841 browser details YourSeq 704 2056 2864 3000 94.2% chrX - 101196714 101197544 831 browser details YourSeq 704 2068 2882 3000 94.4% chr2 - 145721613 145722442 830 browser details YourSeq 704 2058 2864 3000 95.0% chr2 - 77667617 77668465 849 browser details YourSeq 703 2060 2864 3000 94.2% chr3 - 148050524 148051348 825 browser details YourSeq 703 2061 2864 3000 94.1% chr7 + 137890950 137891772 823 browser details YourSeq 701 2062 2864 3000 93.9% chr14 - 29774411 29775233 823 browser details YourSeq 699 2055 2864 3000 94.2% chr6 - 135864928 135865759 832 browser details YourSeq 699 2058 2863 3000 93.8% chr12 + 73418227 73419044 818 browser details YourSeq 698 2051 2864 3000 93.8% chr2 - 162880930 162881775 846 browser details YourSeq 697 2057 2864 3000 93.7% chr10 - 88034558 88035390 833 browser details YourSeq 696 2059 2864 3000 93.6% chr13 + 83851498 83852332 835 browser details YourSeq 695 2061 2864 3000 93.6% chr2 - 154094949 154095776 828 browser details YourSeq 694 2074 2864 3000 94.2% chr9 + 84414641 84415452 812 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% chr3 + 109497205 109500204 3000 browser details YourSeq 54 33 550 3000 67.8% chr7 - 80792883 80793178 296 browser details YourSeq 53 490 554 3000 90.8% chr19 - 41047973 41048037 65 browser details YourSeq 51 506 636 3000 79.7% chr5 - 136639430 136639544 115 browser details YourSeq 51 493 553 3000 91.9% chr16 - 94308870 94308930 61 browser details YourSeq 50 429 551 3000 88.0% chr13 - 12698369 12698489 121 browser details YourSeq 50 429 551 3000 88.0% chr13 - 13433703 13433823 121 browser details YourSeq 45 507 638 3000 73.5% chr4 + 57090583 57090676 94 browser details YourSeq 43 510 561 3000 92.4% chr10 + 65391171 65391635 465 browser details YourSeq 41 508 560 3000 88.7% chr3 - 32115380 32115432 53 browser details YourSeq 41 537 667 3000 73.5% chr17 - 70604738 70604855 118 browser details YourSeq 41 1784 1889 3000 95.8% chr12 + 52301346 52301786 441 browser details YourSeq 40 506 791 3000 95.5% chr11 + 57715113 57715398 286 browser details YourSeq 39 506 550 3000 93.4% chr12 + 76723554 76723598 45 browser details YourSeq 38 506 547 3000 95.3% chr8 - 83806874 83806915 42 browser details YourSeq 38 506 553 3000 89.6% chr10 - 86083491 86083538 48 browser details YourSeq 37 511 553 3000 93.1% chr15 - 58239130 58239172 43 browser details YourSeq 37 507 547 3000 95.2% chr1 - 72792998 72793038 41 browser details YourSeq 37 508 550 3000 93.1% chr12 + 12492522 12492564 43 browser details YourSeq 36 507 550 3000 86.1% chr5 - 138691529 138691571 43 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Vav3 vav 3 oncogene [ Mus musculus (house mouse) ] Gene ID: 57257, updated on 22-Oct-2019 Gene summary Official Symbol Vav3 provided by MGI Official Full Name vav 3 oncogene provided by MGI Primary source MGI:MGI:1888518 See related Ensembl:ENSMUSG00000033721 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 Idd18.1; AA986410; A530094I06Rik Expression Ubiquitous expression in whole brain E14.5 (RPKM 2.7), CNS E14 (RPKM 2.5) and 27 other tissues See more Orthologs human all Genomic context Location: 3; 3 F3 See Vav3 in Genome Data Viewer Exon count: 28 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (109340317..109685694) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (109143601..109488612) Chromosome 3 - NC_000069.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Vav3 ENSMUSG00000033721 Description vav 3 oncogene [Source:MGI Symbol;Acc:MGI:1888518] Gene Synonyms A530094I06Rik, Idd18.1 Location Chromosome 3: 109,340,653-109,685,698 forward strand. GRCm38:CM000996.2 About this gene This gene has 6 transcripts (splice variants), 275 orthologues, 20 paralogues, is a member of 1 Ensembl protein family and is associated with 29 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Vav3-201 ENSMUST00000046864.13 5019 847aa ENSMUSP00000036270.7 Protein coding CCDS17773 Q9R0C8 TSL:1 GENCODE basic APPRIS P1 Vav3-202 ENSMUST00000106576.2 3123 287aa ENSMUSP00000102186.2 Protein coding CCDS59650 Q9R0C8 TSL:1 GENCODE basic Vav3-203 ENSMUST00000126179.7 2398 No protein - Retained intron - - TSL:1 Vav3-206 ENSMUST00000148866.1 579 No protein - lncRNA - - TSL:3 Vav3-204 ENSMUST00000128033.1 515 No protein - lncRNA - - TSL:3 Vav3-205 ENSMUST00000137871.1 465 No protein - lncRNA - - TSL:1 365.05 kb Forward strand 109.4Mb 109.5Mb 109.6Mb Genes (Comprehensive set... Vav3-201 >protein coding Vav3-203 >retained intron Vav3-202 >protein coding Vav3-205 >lncRNA Vav3-204 >lncRNA Vav3-206 >lncRNA Contigs AL671857.16 > AL731716.10 > AL671987.12 > Regulatory Build 109.4Mb 109.5Mb 109.6Mb Reverse strand 365.05 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 RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000046864 345.05 kb Forward strand Vav3-201 >protein coding ENSMUSP00000036... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Dbl homology (DH) domain superfamily SH2 domain superfamily CH domain superfamily SSF50729 SH3-like domain superfamily SMART Calponin homology domain Dbl homology (DH) domain Pleckstrin homology domain SH2 domain Protein kinase C-like, phorbol ester/diacylglycerol-binding domain SH3 domain Prints Smooth muscle protein/calponin SH2 domain SH3 domain Pfam Dbl homology (DH) domain Pleckstrin homology domain SH3 domain SH3 domain Calmodulin-regulated
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