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Mouse Nol12 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Nol12 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Nol12 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Nol12 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nol12 gene (NCBI Reference Sequence: NM_133800 ; Ensembl: ENSMUSG00000033099 ) is located on Mouse chromosome 15. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000138880). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nol12 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-350J4 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: Exon 2 starts from about 13.36% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1403 bp, and the size of intron 3 for 3'-loxP site insertion: 625 bp. The size of effective cKO region: ~1204 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 2 3 4 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Nol12 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(7704bp) | A(25.14% 1937) | C(24.57% 1893) | T(24.01% 1850) | G(26.27% 2024) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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% chr15 + 78933230 78936229 3000 browser details YourSeq 225 901 1314 3000 93.8% chr8 + 89080202 89080616 415 browser details YourSeq 225 400 1314 3000 90.6% chr2 + 112400621 112401491 871 browser details YourSeq 218 400 1074 3000 87.9% chr6 - 148948159 148948546 388 browser details YourSeq 218 901 1314 3000 94.1% chr17 + 66103586 66104145 560 browser details YourSeq 218 901 1357 3000 96.3% chr10 + 62918109 62933565 15457 browser details YourSeq 207 901 1302 3000 87.7% chr11 - 4698547 4698873 327 browser details YourSeq 207 904 1302 3000 87.1% chr10 + 95732840 95733168 329 browser details YourSeq 206 454 1298 3000 92.7% chr11 + 62416767 62417782 1016 browser details YourSeq 202 419 1075 3000 88.8% chr11 - 77545848 77546339 492 browser details YourSeq 200 901 1302 3000 89.9% chr11 + 57994011 57994265 255 browser details YourSeq 195 901 1279 3000 95.9% chr7 + 30128914 30129591 678 browser details YourSeq 192 797 1079 3000 97.6% chr10 - 23814918 23815265 348 browser details YourSeq 187 901 1259 3000 94.8% chr11 - 98988105 98988663 559 browser details YourSeq 183 453 1075 3000 92.4% chr11 - 51991702 51992301 600 browser details YourSeq 181 453 1075 3000 87.5% chr15 - 73220236 73220522 287 browser details YourSeq 180 901 1259 3000 88.6% chr13 - 74516399 74516733 335 browser details YourSeq 178 871 1077 3000 94.2% chr8 - 25211166 25211491 326 browser details YourSeq 176 900 1076 3000 100.0% chr8 + 122616985 122617163 179 browser details YourSeq 174 901 1075 3000 100.0% chr2 + 180946393 180946569 177 Note: The 3000 bp section upstream of Exon 2 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% chr15 + 78937434 78940433 3000 browser details YourSeq 141 916 1232 3000 81.3% chr2 - 181399726 181399939 214 browser details YourSeq 136 1059 1347 3000 90.5% chr14 + 20856480 20857343 864 browser details YourSeq 135 1039 1232 3000 92.5% chr12 - 54853267 54853521 255 browser details YourSeq 134 1056 1229 3000 85.6% chr1 - 37257376 37257541 166 browser details YourSeq 132 1054 1326 3000 87.6% chr4 + 126078274 126078623 350 browser details YourSeq 131 1054 1232 3000 88.4% chr5 + 89488876 89489068 193 browser details YourSeq 131 1055 1217 3000 91.5% chr14 + 70481890 70737887 255998 browser details YourSeq 127 1054 1217 3000 89.1% chr18 - 7665096 7665263 168 browser details YourSeq 127 1060 1231 3000 88.5% chr12 + 110526170 110526341 172 browser details YourSeq 127 1054 1216 3000 87.5% chr12 + 20897027 20897187 161 browser details YourSeq 126 1055 1217 3000 89.6% chr11 - 32365412 32365600 189 browser details YourSeq 126 1060 1235 3000 85.9% chr1 - 86458654 86458815 162 browser details YourSeq 125 1056 1214 3000 90.0% chr8 + 73050679 73050840 162 browser details YourSeq 125 1053 1214 3000 88.9% chr6 + 74036086 74036247 162 browser details YourSeq 124 1025 1217 3000 84.8% chr15 - 10507811 10508021 211 browser details YourSeq 124 1061 1231 3000 84.4% chr15 + 102260591 102260752 162 browser details YourSeq 124 1057 1217 3000 90.8% chr1 + 171133696 171133905 210 browser details YourSeq 123 1055 1217 3000 86.3% chr11 - 3874300 3874456 157 browser details YourSeq 122 1056 1226 3000 85.9% chr8 - 75063965 75064131 167 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: Nol12 nucleolar protein 12 [ Mus musculus (house mouse) ] Gene ID: 97961, updated on 12-Aug-2019 Gene summary Official Symbol Nol12 provided by MGI Official Full Name nucleolar protein 12 provided by MGI Primary source MGI:MGI:2146285 See related Ensembl:ENSMUSG00000033099 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 Nop25; C78541 Expression Ubiquitous expression in liver E14 (RPKM 7.9), CNS E11.5 (RPKM 7.9) and 28 other tissues See more Orthologs human all Genomic context Location: 15; 15 E1 See Nol12 in Genome Data Viewer Exon count: 8 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (78934933..78941910) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (78765363..78772340) Chromosome 15 - NC_000081.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Nol12 ENSMUSG00000033099 Description nucleolar protein 12 [Source:MGI Symbol;Acc:MGI:2146285] Gene Synonyms C78541, Nop25 Location Chromosome 15: 78,934,933-78,943,638 forward strand. GRCm38:CM001008.2 About this gene This gene has 4 transcripts (splice variants), 190 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nol12-203 ENSMUST00000138880.8 3721 217aa ENSMUSP00000116103.1 Protein coding CCDS27629 Q8BG17 TSL:1 GENCODE basic APPRIS P1 Nol12-201 ENSMUST00000041164.3 742 195aa ENSMUSP00000042908.3 Protein coding - F8WGP0 CDS 5' incomplete TSL:3 Nol12-202 ENSMUST00000123013.1 681 83aa ENSMUSP00000121877.1 Protein coding - B7ZCM5 CDS 3' incomplete TSL:5 Nol12-204 ENSMUST00000145157.1 681 15aa ENSMUSP00000122905.1 Protein coding - B7ZBY4 CDS 3' incomplete TSL:5 28.71 kb Forward strand 78.93Mb 78.94Mb 78.95Mb Genes (Comprehensive set... Lgals1-201 >protein coding Nol12-203 >protein coding Triobp-204 >protein coding Lgals1-202 >nonsense mediated decay Nol12-201 >protein coding Triobp-203 >protein coding Nol12-204 >protein coding Triobp-208 >protein coding Nol12-202 >protein coding Triobp-212 >protein coding Triobp-207 >retained intron Contigs < AL831713.4 < AL589670.21 Regulatory Build 78.93Mb 78.94Mb 78.95Mb Reverse strand 28.71 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000138880 8.71 kb Forward strand Nol12-203 >protein coding ENSMUSP00000116... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Nucleolar protein 12 PANTHER Nucleolar protein 12 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 217 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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