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Mouse Hsd17b3 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Hsd17b3 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Hsd17b3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Hsd17b3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Hsd17b3 gene (NCBI Reference Sequence: NM_008291 ; Ensembl: ENSMUSG00000033122 ) is located on Mouse chromosome 13. 11 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 11 (Transcript: ENSMUST00000166224). 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 Hsd17b3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-238F4 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 3 starts from about 20.77% 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: 10747 bp, and the size of intron 3 for 3'-loxP site insertion: 2403 bp. The size of effective cKO region: ~576 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 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Hsd17b3 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(7076bp) | A(26.29% 1860) | C(20.58% 1456) | T(31.15% 2204) | G(21.99% 1556) 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% chr13 - 64076674 64079673 3000 browser details YourSeq 168 1292 1953 3000 87.4% chr1 - 4757905 5000202 242298 browser details YourSeq 121 1298 1930 3000 89.6% chr11 + 101693611 101711697 18087 browser details YourSeq 107 1287 1417 3000 93.6% chrX - 36679685 36679819 135 browser details YourSeq 107 1254 1429 3000 84.4% chr15 + 89930246 89930403 158 browser details YourSeq 97 1137 1502 3000 93.0% chr7 + 143889451 143889977 527 browser details YourSeq 96 1287 1435 3000 90.7% chr13 + 89351446 89351604 159 browser details YourSeq 91 1297 1428 3000 90.4% chr2 - 154762604 154762733 130 browser details YourSeq 90 1304 1422 3000 90.0% chr8 + 94119541 94119655 115 browser details YourSeq 88 1298 1428 3000 88.5% chr11 + 30400321 30400448 128 browser details YourSeq 86 1442 1958 3000 74.6% chr14 + 30193569 30193977 409 browser details YourSeq 85 1292 1422 3000 89.5% chr12 + 3564117 3564246 130 browser details YourSeq 83 1292 1427 3000 90.4% chr1 - 63385842 63385978 137 browser details YourSeq 79 1304 1603 3000 71.9% chr2 + 157156454 157156565 112 browser details YourSeq 79 1277 1389 3000 91.9% chr1 + 180543639 180543754 116 browser details YourSeq 78 1445 1923 3000 72.9% chr11 + 73163533 73163886 354 browser details YourSeq 75 1760 1945 3000 78.5% chr5 - 63997465 63997651 187 browser details YourSeq 74 1304 1426 3000 91.7% chr11 + 64380634 64380754 121 browser details YourSeq 74 1292 1422 3000 92.1% chr10 + 55387568 55387698 131 browser details YourSeq 72 1760 1948 3000 85.8% chr6 - 30894336 30894523 188 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% chr13 - 64073098 64076097 3000 browser details YourSeq 381 131 803 3000 88.4% chr1 - 30878730 30879330 601 browser details YourSeq 371 136 795 3000 88.7% chr11 - 101817860 101818396 537 browser details YourSeq 352 402 803 3000 93.2% chr4 + 133909240 133909636 397 browser details YourSeq 349 139 803 3000 88.9% chr10 - 70137061 70137495 435 browser details YourSeq 345 403 817 3000 91.1% chrX - 51312768 51313176 409 browser details YourSeq 345 395 803 3000 92.8% chr5 - 84004541 84004953 413 browser details YourSeq 344 400 817 3000 92.4% chr5 - 28804597 28805014 418 browser details YourSeq 343 400 803 3000 91.9% chrX - 163797931 163798328 398 browser details YourSeq 342 401 805 3000 91.9% chr9 - 51015153 51015552 400 browser details YourSeq 342 402 795 3000 92.1% chr2 - 22706961 22707348 388 browser details YourSeq 341 416 803 3000 93.5% chr3 - 153558527 153558911 385 browser details YourSeq 341 402 805 3000 92.5% chr18 + 4269116 4269514 399 browser details YourSeq 340 403 803 3000 91.9% chrX - 166108986 166109383 398 browser details YourSeq 340 400 803 3000 91.6% chrX - 157514826 157515219 394 browser details YourSeq 340 413 803 3000 92.5% chr4 - 145327221 145327607 387 browser details YourSeq 340 394 803 3000 92.9% chr7 + 132590105 132590552 448 browser details YourSeq 340 403 803 3000 91.4% chr6 + 128589883 128590277 395 browser details YourSeq 339 404 803 3000 92.1% chr5 - 37075482 37075878 397 browser details YourSeq 339 402 803 3000 91.5% chrX + 6012619 6013006 388 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: Hsd17b3 hydroxysteroid (17-beta) dehydrogenase 3 [ Mus musculus (house mouse) ] Gene ID: 15487, updated on 15-Oct-2019 Gene summary Official Symbol Hsd17b3 provided by MGI Official Full Name hydroxysteroid (17-beta) dehydrogenase 3 provided by MGI Primary source MGI:MGI:107177 See related Ensembl:ENSMUSG00000033122 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 Expression Restricted expression toward testis adult (RPKM 6.2) See more Orthologs human all Genomic context Location: 13; 13 B3 See Hsd17b3 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (64058274..64089262, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (64159582..64190509, complement) Chromosome 13 - NC_000079.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Hsd17b3 ENSMUSG00000033122 Description hydroxysteroid (17-beta) dehydrogenase 3 [Source:MGI Symbol;Acc:MGI:107177] Gene Synonyms 17(beta)HSD type 3 Location Chromosome 13: 64,058,266-64,089,230 reverse strand. GRCm38:CM001006.2 About this gene This gene has 5 transcripts (splice variants), 178 orthologues, 31 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Hsd17b3-202 ENSMUST00000166224.7 1286 305aa ENSMUSP00000132011.1 Protein coding CCDS26594 P70385 TSL:2 GENCODE basic APPRIS P1 Hsd17b3-204 ENSMUST00000222783.1 1184 305aa ENSMUSP00000152848.1 Protein coding CCDS26594 P70385 TSL:5 GENCODE basic APPRIS P1 Hsd17b3-201 ENSMUST00000039832.6 1131 305aa ENSMUSP00000044217.6 Protein coding CCDS26594 P70385 TSL:1 GENCODE basic APPRIS P1 Hsd17b3-203 ENSMUST00000221513.1 655 123aa ENSMUSP00000152478.1 Protein coding - A0A1Y7VJL6 CDS 5' incomplete TSL:5 Hsd17b3-205 ENSMUST00000222810.1 653 193aa ENSMUSP00000152274.1 Protein coding - A0A1Y7VJ36 CDS 3' incomplete TSL:3 50.97 kb Forward strand 64.05Mb 64.06Mb 64.07Mb 64.08Mb 64.09Mb Contigs CT009717.9 > Genes (Comprehensive set... < Hsd17b3-204protein coding < Slc35d2-203retained intron < Hsd17b3-202protein coding < Slc35d2-201protein coding < Hsd17b3-201protein coding < Slc35d2-202protein coding < Hsd17b3-203protein coding < Slc35d2-208nonsense mediated decay < Hsd17b3-205protein coding < Slc35d2-206nonsense mediated decay Regulatory Build 64.05Mb 64.06Mb 64.07Mb 64.08Mb 64.09Mb Reverse strand 50.97 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000166224 < Hsd17b3-202protein coding Reverse strand 30.93 kb ENSMUSP00000132... Superfamily NAD(P)-binding domain superfamily Prints Short-chain dehydrogenase/reductase SDR Short-chain dehydrogenase/reductase SDR Pfam Short-chain dehydrogenase/reductase SDR PROSITE patterns Short-chain dehydrogenase/reductase, conserved site PIRSF PIRSF000126 PANTHER PTHR43899 Testosterone 17-beta-dehydrogenase 3 Gene3D 3.40.50.720 CDD cd05356 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 305 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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