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Mouse Btg3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Btg3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Btg3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Btg3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Btg3 gene (NCBI Reference Sequence: NM_009770 ; Ensembl: ENSMUSG00000022863 ) is located on Mouse chromosome 16. 5 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 5 (Transcript: ENSMUST00000023570). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Btg3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-122K1 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 increased incidence of lung tumors. Exon 4 starts from about 41.27% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 4103 bp, and the size of intron 4 for 3'-loxP site insertion: 4263 bp. The size of effective cKO region: ~708 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 4 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Btg3 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(7208bp) | A(30.62% 2207) | C(19.12% 1378) | T(30.83% 2222) | G(19.44% 1401) 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% chr16 - 78365260 78368259 3000 browser details YourSeq 372 513 1034 3000 95.4% chr18 - 24829776 24830537 762 browser details YourSeq 367 515 909 3000 96.5% chr13 + 39514565 39514959 395 browser details YourSeq 366 515 908 3000 96.5% chr3 - 102904587 102904980 394 browser details YourSeq 366 515 908 3000 96.5% chr3 - 102906071 102906464 394 browser details YourSeq 366 515 908 3000 96.5% chr5 + 108493138 108493531 394 browser details YourSeq 366 514 915 3000 95.8% chr18 + 29113473 29113875 403 browser details YourSeq 366 515 908 3000 96.5% chr18 + 12713555 12713948 394 browser details YourSeq 365 512 909 3000 96.0% chr6 - 24143299 24143696 398 browser details YourSeq 365 515 909 3000 96.3% chr3 - 105332089 105332483 395 browser details YourSeq 365 514 908 3000 96.5% chr17 - 51851183 51851579 397 browser details YourSeq 365 515 908 3000 96.5% chr2 + 27787519 27787913 395 browser details YourSeq 365 515 906 3000 96.7% chr2 + 6132463 6132855 393 browser details YourSeq 365 515 909 3000 96.3% chr1 + 151744297 151744691 395 browser details YourSeq 365 515 921 3000 94.8% chr1 + 23236366 23236770 405 browser details YourSeq 364 513 910 3000 95.3% chr4 - 44946795 44947191 397 browser details YourSeq 364 489 908 3000 93.3% chr19 - 24240116 24240519 404 browser details YourSeq 364 513 907 3000 96.3% chr16 - 28492330 28492725 396 browser details YourSeq 364 515 909 3000 96.3% chr14 - 24417487 24417882 396 browser details YourSeq 364 514 909 3000 96.0% chr5 + 141460981 141461376 396 Note: The 3000 bp section upstream of Exon 4 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% chr16 - 78361552 78364551 3000 browser details YourSeq 285 421 2195 3000 91.1% chr10 + 127029801 127681822 652022 browser details YourSeq 246 443 2183 3000 91.3% chr15 - 84876635 85103956 227322 browser details YourSeq 158 421 992 3000 82.7% chr11 + 95816857 95817114 258 browser details YourSeq 149 338 581 3000 91.2% chr11 - 94908667 94909138 472 browser details YourSeq 149 410 590 3000 91.0% chr15 + 20847942 20848121 180 browser details YourSeq 148 338 577 3000 88.8% chr11 - 88900428 88900666 239 browser details YourSeq 145 410 590 3000 90.1% chr6 - 28102241 28102421 181 browser details YourSeq 145 411 590 3000 91.0% chr5 + 117073305 117073488 184 browser details YourSeq 144 2028 2183 3000 96.2% chr4 + 150922842 150922997 156 browser details YourSeq 143 410 581 3000 92.4% chr12 - 4903733 4903909 177 browser details YourSeq 143 410 597 3000 88.7% chr1 - 192199958 192200148 191 browser details YourSeq 143 409 591 3000 90.0% chr1 + 180319603 180596458 276856 browser details YourSeq 142 1656 2172 3000 83.4% chr1 - 135211735 135212113 379 browser details YourSeq 142 409 591 3000 89.1% chr12 + 87423103 87423293 191 browser details YourSeq 142 421 590 3000 91.8% chr11 + 29695693 29695862 170 browser details YourSeq 141 408 581 3000 91.8% chr17 - 27670769 27670951 183 browser details YourSeq 140 411 581 3000 89.4% chr11 - 97266793 97266961 169 browser details YourSeq 140 410 581 3000 92.2% chrX + 153222620 153222791 172 browser details YourSeq 140 410 581 3000 91.7% chr15 + 77361088 77361265 178 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: Btg3 BTG anti-proliferation factor 3 [ Mus musculus (house mouse) ] Gene ID: 12228, updated on 27-Aug-2019 Gene summary Official Symbol Btg3 provided by MGI Official Full Name BTG anti-proliferation factor 3 provided by MGI Primary source MGI:MGI:109532 See related Ensembl:ENSMUSG00000022863 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 ANA; tob5 Summary This gene encodes B cell translocation gene 3, a member of the BTG gene family. This family is defined by a conserved N- Expression terminal domain, known to bind transcription factors, and a less conserved C-terminal domain. This protein is thought to have anti-proliferative properties, and may be involved in regulating the G1-S transition to suppress cell cycle progression. Mice deficient for this gene display an increased incidence of lung cancers, and many human lung cancer cells exhibit decreased levels of B cell translocation gene 3. Alternate splicing results in multiple transcript variants. A pseudogene of this gene is found on chromosome 17. [provided by RefSeq, Jul 2014] Orthologs Broad expression in genital fat pad adult (RPKM 36.7), placenta adult (RPKM 14.6) and 20 other tissues See more human all Genomic context Location: 16; 16 C3.1 See Btg3 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (78359860..78377181, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (78360105..78377030, complement) Chromosome 16 - NC_000082.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Btg3 ENSMUSG00000022863 Description B cell translocation gene 3 [Source:MGI Symbol;Acc:MGI:109532] Gene Synonyms ANA, tob5 Location Chromosome 16: 78,332,637-78,377,192 reverse strand. GRCm38:CM001009.2 About this gene This gene has 3 transcripts (splice variants), 213 orthologues, 5 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Btg3-201 ENSMUST00000023570.13 1414 252aa ENSMUSP00000023570.7 Protein coding CCDS28277 P50615 Q52L83 TSL:1 GENCODE basic APPRIS P1 Btg3-203 ENSMUST00000231353.1 3013 152aa ENSMUSP00000156138.1 Protein coding - A0A338P787 CDS 5' incomplete Btg3-202 ENSMUST00000148124.1 442 137aa ENSMUSP00000119706.1 Protein coding - D3Z1M9 CDS 3' incomplete TSL:5 64.56 kb Forward strand 78.33Mb 78.34Mb 78.35Mb 78.36Mb 78.37Mb 78.38Mb Genes Cxadr-205 >protein coding (Comprehensive set... Cxadr-201 >protein coding Cxadr-202 >protein coding Cxadr-204 >protein coding Cxadr-206 >retained intron Cxadr-203 >nonsense mediated decay Contigs AC130844.3 > Genes (Comprehensive set... < Btg3-203protein coding < Gm49590-201TEC < Btg3-201protein coding < Btg3-202protein coding Regulatory Build 78.33Mb 78.34Mb 78.35Mb 78.36Mb 78.37Mb 78.38Mb Reverse strand 64.56 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 Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000023570 < Btg3-201protein coding Reverse strand 16.95 kb ENSMUSP00000023... MobiDB lite Low complexity (Seg) Superfamily BTG-like domain superfamily SMART Anti-proliferative protein Prints Anti-proliferative protein Pfam Anti-proliferative protein PROSITE patterns Anti-proliferative protein Anti-proliferative protein PANTHER PTHR22978 PTHR22978:SF6 Gene3D BTG-like domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 252 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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