https://www.alphaknockout.com

Mouse Tnfaip8 Knockout Project (CRISPR/Cas9)

Objective: To create a Tnfaip8 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tnfaip8 gene (NCBI Reference Sequence: NM_134131 ; Ensembl: ENSMUSG00000062210 ) is located on Mouse chromosome 18. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000148159). Exon 2 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit increased susceptibility to induced colitis.

Exon 2 starts from about 11.64% of the coding region. Exon 2 covers 88.52% of the coding region. The size of effective KO region: ~563 bp. The KO 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 2

Legends Exon of mouse Tnfaip8 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 563 bp section of Exon 2 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 563 bp section of Exon 2 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(563bp) | A(29.48% 166) | C(25.58% 144) | T(20.96% 118) | G(23.98% 135)

Note: The 563 bp section of Exon 2 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(563bp) | A(29.48% 166) | C(25.58% 144) | T(20.96% 118) | G(23.98% 135)

Note: The 563 bp section of Exon 2 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 563 1 563 563 100.0% chr18 + 50090202 50090764 563 browser details YourSeq 48 46 133 563 77.3% chr9 - 54027506 54027593 88 browser details YourSeq 29 267 332 563 91.5% chr14 + 94032205 94032632 428 browser details YourSeq 25 477 505 563 80.8% chrX - 109515806 109515831 26 browser details YourSeq 22 120 145 563 84.0% chr18 - 59025522 59025546 25 browser details YourSeq 21 477 497 563 100.0% chr10 + 64159705 64159725 21 browser details YourSeq 20 494 513 563 100.0% chr16 - 58937174 58937193 20 browser details YourSeq 20 346 365 563 100.0% chr16 + 70689493 70689512 20

Note: The 563 bp section 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 563 1 563 563 100.0% chr18 + 50090200 50090762 563 browser details YourSeq 53 297 374 563 85.4% chr17 + 56172003 56172080 78 browser details YourSeq 48 48 135 563 77.3% chr9 - 54027506 54027593 88 browser details YourSeq 25 479 507 563 80.8% chrX - 109515806 109515831 26 browser details YourSeq 22 122 147 563 84.0% chr18 - 59025522 59025546 25 browser details YourSeq 21 479 499 563 100.0% chr10 + 64159705 64159725 21 browser details YourSeq 20 496 515 563 100.0% chr16 - 58937174 58937193 20 browser details YourSeq 20 348 367 563 100.0% chr16 + 70689493 70689512 20

Note: The 563 bp section of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tnfaip8 tumor necrosis factor, alpha-induced protein 8 [ Mus musculus (house mouse) ] Gene ID: 106869, updated on 12-Aug-2019

Gene summary

Official Symbol Tnfaip8 provided by MGI Official Full Name tumor necrosis factor, alpha-induced protein 8 provided by MGI Primary source MGI:MGI:2147191 See related Ensembl:ENSMUSG00000062210 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 Nded; Tipe; Gg2-1; Ssc-2; Gm10539; AA987150; E130304C20Rik Expression Broad expression in kidney adult (RPKM 36.9), placenta adult (RPKM 10.3) and 15 other tissues See more Orthologs human all

Genomic context

Location: 18; 18 D1 See Tnfaip8 in Genome Data Viewer Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (49979427..50093229)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (50139081..50252883)

Chromosome 18 - NC_000084.6

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Transcript information: This gene has 13 transcripts

Gene: Tnfaip8 ENSMUSG00000062210

Description tumor necrosis factor, alpha-induced protein 8 [Source:MGI Symbol;Acc:MGI:2147191] Gene Synonyms E130304C20Rik, Gg2-1, Gm10539, Nded, Ssc-2, Tipe Location Chromosome 18: 49,979,427-50,107,173 forward strand. GRCm38:CM001011.2 About this gene This gene has 13 transcripts (splice variants), 140 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tnfaip8-212 ENSMUST00000179937.1 2385 136aa ENSMUSP00000136030.1 Protein coding CCDS50287 Q921Z5 TSL:1 GENCODE basic

Tnfaip8-207 ENSMUST00000148159.2 1921 212aa ENSMUSP00000119041.2 Protein coding CCDS37817 D3Z325 TSL:1 GENCODE basic

Tnfaip8-208 ENSMUST00000148989.2 1002 188aa ENSMUSP00000120712.2 Protein coding CCDS50288 D3YWJ5 TSL:2 GENCODE basic APPRIS P1

Tnfaip8-203 ENSMUST00000126666.2 906 212aa ENSMUSP00000121372.2 Protein coding - D3Z312 TSL:2 GENCODE basic

Tnfaip8-204 ENSMUST00000128377.1 822 166aa ENSMUSP00000136152.1 Protein coding - J3QM73 CDS 3' incomplete TSL:2

Tnfaip8-210 ENSMUST00000153873.2 784 114aa ENSMUSP00000115396.2 Protein coding - D3Z0Q1 CDS 3' incomplete TSL:5

Tnfaip8-205 ENSMUST00000134348.7 658 77aa ENSMUSP00000119533.1 Protein coding - D3Z205 CDS 3' incomplete TSL:3

Tnfaip8-206 ENSMUST00000145726.1 632 100aa ENSMUSP00000136665.1 Protein coding - J3QNA5 TSL:3 GENCODE basic

Tnfaip8-213 ENSMUST00000180305.1 369 122aa ENSMUSP00000136682.1 Protein coding - J3QNB8 TSL:5 GENCODE basic

Tnfaip8-211 ENSMUST00000155403.1 790 No protein - Retained intron - - TSL:3

Tnfaip8-201 ENSMUST00000087655.5 5085 No protein - lncRNA - - TSL:5

Tnfaip8-209 ENSMUST00000153545.1 528 No protein - lncRNA - - TSL:2

Tnfaip8-202 ENSMUST00000124175.1 392 No protein - lncRNA - - TSL:3

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147.75 kb Forward strand

50.00Mb 50.05Mb 50.10Mb Genes (Comprehensive set... Tnfaip8-208 >protein coding

Tnfaip8-201 >lncRNA Tnfaip8-211 >retained intron

Tnfaip8-212 >protein coding

Tnfaip8-209 >lncRNA

Tnfaip8-203 >protein coding

Tnfaip8-206 >protein coding

Tnfaip8-202 >lncRNA Tnfaip8-205 >protein coding

Tnfaip8-204 >protein coding

Tnfaip8-213 >protein coding

Tnfaip8-210 >protein coding

Tnfaip8-207 >protein coding

Contigs < AC166570.2 AC120859.12 > Genes < C030005K06Rik-203lncRNA (Comprehensive set...

< C030005K06Rik-201lncRNA

< C030005K06Rik-204lncRNA

< C030005K06Rik-202lncRNA

Regulatory Build

50.00Mb 50.05Mb 50.10Mb Reverse strand 147.75 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 RNA gene

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Transcript: ENSMUST00000148159

38.50 kb Forward strand

Tnfaip8-207 >protein coding

ENSMUSP00000119... Coiled-coils (Ncoils) Pfam Tumor necrosis factor alpha-induced protein 8-like PANTHER Tumor necrosis factor alpha-induced protein 8-like

PTHR12757:SF3 Gene3D TNFAIP8 superfamily

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

Variant Legend

missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 212

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

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