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Mouse Ostf1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ostf1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ostf1 gene (NCBI Reference Sequence: NM_017375 ; Ensembl: ENSMUSG00000024725 ) is located on Mouse chromosome 19. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000025631). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ostf1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-247E15 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 knockout results in increased trabecular number and bone density in the femur and tibia.

Exon 4 starts from about 20.62% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2431 bp, and the size of intron 5 for 3'-loxP site insertion: 2074 bp. The size of effective cKO region: ~1837 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ostf1 Homology arm cKO region loxP site

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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(8337bp) | A(27.56% 2298) | C(20.44% 1704) | T(29.85% 2489) | G(22.14% 1846)

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.

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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 - 18594170 18597169 3000 browser details YourSeq 43 224 547 3000 58.7% chr18 - 19743100 19743161 62 browser details YourSeq 42 520 670 3000 97.8% chr14 + 33937741 33937893 153 browser details YourSeq 41 1503 1544 3000 100.0% chr4 - 153390486 153390847 362 browser details YourSeq 41 2438 2491 3000 93.7% chr1 + 118785324 118785441 118 browser details YourSeq 40 1503 1542 3000 100.0% chr4 - 52768807 52768846 40 browser details YourSeq 40 1501 1541 3000 100.0% chr18 - 5386590 5386658 69 browser details YourSeq 40 1505 1544 3000 100.0% chr4 + 52860282 52860321 40 browser details YourSeq 39 1503 1541 3000 100.0% chr5 - 118183548 118183586 39 browser details YourSeq 39 1503 1541 3000 100.0% chr2 - 165265829 165265867 39 browser details YourSeq 39 2438 2494 3000 93.4% chr14 - 32144915 32144973 59 browser details YourSeq 39 1501 1541 3000 97.6% chr17 + 71460899 71460939 41 browser details YourSeq 39 1503 1541 3000 100.0% chr14 + 117269305 117269343 39 browser details YourSeq 38 1503 1541 3000 100.0% chr10 + 81629105 81629147 43 browser details YourSeq 38 1503 1541 3000 100.0% chr10 + 79641447 79641509 63 browser details YourSeq 37 1503 1544 3000 95.3% chr8 - 78248382 78248427 46 browser details YourSeq 37 1505 1541 3000 100.0% chr4 - 150762387 150762423 37 browser details YourSeq 37 1503 1541 3000 97.5% chr19 - 35200787 35200825 39 browser details YourSeq 37 1503 1541 3000 100.0% chr15 - 12678294 12678343 50 browser details YourSeq 37 1503 1541 3000 97.5% chr19 + 25372066 25372104 39

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% chr19 - 18589333 18592332 3000 browser details YourSeq 44 349 445 3000 96.1% chr1 - 23777760 23777857 98 browser details YourSeq 44 65 205 3000 92.0% chr15 + 39326919 39327068 150 browser details YourSeq 43 427 626 3000 93.9% chr13 - 118175400 118175638 239 browser details YourSeq 41 71 364 3000 90.4% chrX + 151884058 151884354 297 browser details YourSeq 41 179 364 3000 70.3% chr3 + 87633070 87633219 150 browser details YourSeq 40 197 364 3000 93.5% chr11 + 70479011 70479187 177 browser details YourSeq 38 427 552 3000 95.3% chr2 + 124914431 124914561 131 browser details YourSeq 35 2391 2437 3000 77.8% chr3 - 14933292 14933327 36 browser details YourSeq 34 51 98 3000 80.9% chr2 - 51568660 51568706 47 browser details YourSeq 34 11 100 3000 81.1% chr8 + 45155521 45155605 85 browser details YourSeq 34 197 246 3000 92.7% chr17 + 82594121 82594181 61 browser details YourSeq 31 350 382 3000 97.0% chr14 - 121187566 121187598 33 browser details YourSeq 29 349 383 3000 91.5% chr12 - 39829035 39829069 35 browser details YourSeq 29 589 623 3000 91.5% chr5 + 67992868 67992902 35 browser details YourSeq 29 11 88 3000 87.9% chr12 + 9793056 9793132 77 browser details YourSeq 27 71 108 3000 93.6% chr13 - 14227956 14227994 39 browser details YourSeq 26 595 622 3000 96.5% chr4 - 55211849 55211876 28 browser details YourSeq 26 591 624 3000 88.3% chr15 + 12282665 12282698 34 browser details YourSeq 23 224 249 3000 96.0% chr13 - 83428366 83428391 26

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ostf1 osteoclast stimulating factor 1 [ Mus musculus (house mouse) ] Gene ID: 20409, updated on 12-Aug-2019

Gene summary

Official Symbol Ostf1 provided by MGI Official Full Name osteoclast stimulating factor 1 provided by MGI Primary source MGI:MGI:700012 See related Ensembl:ENSMUSG00000024725 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 SH3P2; Sh3d3; C78236 Expression Ubiquitous expression in placenta adult (RPKM 31.9), adrenal adult (RPKM 21.5) and 27 other tissues See more Orthologs human all

Genomic context

Location: 19 B; 19 13.17 cM See Ostf1 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (18580364..18631813, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (18654854..18706303, complement)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 5 transcripts

Gene: Ostf1 ENSMUSG00000024725

Description osteoclast stimulating factor 1 [Source:MGI Symbol;Acc:MGI:700012] Gene Synonyms C78236, SH3P2, Sh3d3 Location Chromosome 19: 18,516,137-18,631,823 reverse strand. GRCm38:CM001012.2 About this gene This gene has 5 transcripts (splice variants), 201 orthologues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ostf1- ENSMUST00000025631.6 2726 215aa ENSMUSP00000025631.6 Protein coding CCDS37930 Q62422 TSL:1 201 GENCODE basic APPRIS P1

Ostf1- ENSMUST00000236615.1 974 212aa ENSMUSP00000157998.1 Protein coding - A0A494BA97 GENCODE 204 basic

Ostf1- ENSMUST00000236728.1 1322 38aa ENSMUSP00000157475.1 Nonsense mediated - A0A494B943 - 205 decay

Ostf1- ENSMUST00000156908.7 2816 No - Retained intron - - TSL:5 203 protein

Ostf1- ENSMUST00000138860.1 516 No - Retained intron - - TSL:2 202 protein

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135.69 kb Forward strand 18.52Mb 18.54Mb 18.56Mb 18.58Mb 18.60Mb 18.62Mb 18.64Mb Genes Gm8250-201 >processed pseudogene Nmrk1-204 >nonsense mediated decay (Comprehensive set...

Nmrk1-202 >retained intron

Nmrk1-205 >retained intron

Nmrk1-206 >protein coding

Nmrk1-201 >protein coding

Nmrk1-203 >protein coding

Contigs < AC147474.2 Genes (Comprehensive set... < Ostf1-204protein coding

< Ostf1-201protein coding

< Ostf1-205nonsense mediated decay

< Ostf1-203retained intron

< Ostf1-202retained intron

Regulatory Build

18.52Mb 18.54Mb 18.56Mb 18.58Mb 18.60Mb 18.62Mb 18.64Mb Reverse strand 135.69 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

pseudogene processed transcript

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Transcript: ENSMUST00000025631

< Ostf1-201protein coding

Reverse strand 52.46 kb

ENSMUSP00000025... MobiDB lite Low complexity (Seg) Superfamily Ankyrin repeat-containing domain superfamily

SH3-like domain superfamily SMART SH3 domain Ankyrin repeat Prints PR00499 Ankyrin repeat

SH3 domain Pfam SH3 domain Ankyrin repeat-containing domain

PROSITE profiles SH3 domain Ankyrin repeat-containing domain

Ankyrin repeat PANTHER PTHR24155:SF10

PTHR24155 Gene3D 2.30.30.40 Ankyrin repeat-containing domain superfamily

CDD cd11772

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 215

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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