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Mouse Tnfaip1 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Tnfaip1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Tnfaip1 gene (NCBI Reference Sequence: NM_001159392 ; Ensembl: ENSMUSG00000017615 ) is located on Mouse chromosome 11. 7 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000108277). Exon 3~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tnfaip1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-356D12 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 21.73% of the coding region. The knockout of Exon 3~6 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 788 bp, and the size of intron 6 for 3'-loxP site insertion: 1984 bp. The size of effective cKO region: ~2488 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 2 3 4 5 6 7 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Tnfaip1 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.
Overview of the GC Content Distribution Window size: 300 bp
Sequence 12
Summary: Full Length(8714bp) | A(22.94% 1999) | C(25.17% 2193) | T(25.9% 2257) | G(25.99% 2265)
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% chr11 - 78529490 78532489 3000 browser details YourSeq 243 558 985 3000 92.7% chr15 - 73427148 73427762 615 browser details YourSeq 198 622 985 3000 93.1% chr4 + 129632951 129633593 643 browser details YourSeq 180 634 986 3000 93.7% chr1 - 155513166 155513796 631 browser details YourSeq 180 627 985 3000 91.0% chr11 + 106950147 106950488 342 browser details YourSeq 175 370 981 3000 84.7% chr2 + 168267597 168267969 373 browser details YourSeq 172 392 987 3000 83.0% chr11 - 20171489 20171735 247 browser details YourSeq 163 577 985 3000 85.8% chr9 + 108504069 108504282 214 browser details YourSeq 162 381 985 3000 84.3% chr2 + 166838073 166838328 256 browser details YourSeq 161 845 1641 3000 95.0% chr3 + 10296662 10473722 177061 browser details YourSeq 153 564 977 3000 94.2% chr9 - 89089897 89090328 432 browser details YourSeq 151 328 955 3000 83.2% chr2 + 28949512 28949796 285 browser details YourSeq 148 383 985 3000 83.6% chr6 + 115726101 115726432 332 browser details YourSeq 141 556 954 3000 87.4% chr11 - 105144057 105144592 536 browser details YourSeq 141 843 997 3000 96.1% chr7 + 126258235 126258389 155 browser details YourSeq 141 617 985 3000 86.1% chr2 + 90862428 90862727 300 browser details YourSeq 139 833 989 3000 93.0% chr7 - 123072846 123073001 156 browser details YourSeq 139 843 985 3000 98.7% chr4 - 11224720 11224862 143 browser details YourSeq 138 541 708 3000 89.4% chr5 - 86137149 86137309 161 browser details YourSeq 138 844 985 3000 98.6% chr4 - 151797409 151797550 142
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% chr11 - 78524276 78527275 3000 browser details YourSeq 53 2860 2954 3000 90.8% chr4 - 13748832 13748931 100 browser details YourSeq 51 2857 2938 3000 94.9% chr11 - 19902681 19902897 217 browser details YourSeq 51 2863 2943 3000 93.4% chr12 + 86035660 86035759 100 browser details YourSeq 49 2860 2934 3000 93.0% chrX - 60463044 60463118 75 browser details YourSeq 49 2475 2937 3000 61.9% chr18 + 75688380 75688476 97 browser details YourSeq 45 1084 1451 3000 98.0% chr12 - 100245997 100246457 461 browser details YourSeq 42 2897 2958 3000 93.8% chr9 + 50740066 50740127 62 browser details YourSeq 42 2480 2896 3000 55.8% chr12 + 12755992 12756049 58 browser details YourSeq 41 2910 2963 3000 91.9% chr11 - 50962420 50962474 55 browser details YourSeq 41 2857 2937 3000 90.7% chr2 + 164455247 164455325 79 browser details YourSeq 41 2861 2928 3000 92.0% chr13 + 56199165 56199232 68 browser details YourSeq 40 2857 2900 3000 97.8% chr12 - 65218054 65218575 522 browser details YourSeq 39 2852 2928 3000 69.1% chr12 - 59498260 59498302 43 browser details YourSeq 39 2857 2933 3000 90.7% chr7 + 127262966 127263041 76 browser details YourSeq 39 2856 2937 3000 91.5% chr6 + 145834790 145834871 82 browser details YourSeq 38 324 388 3000 78.6% chr18 + 68696526 68696579 54 browser details YourSeq 37 1061 1105 3000 97.5% chr9 + 37590081 37590126 46 browser details YourSeq 36 2857 2937 3000 95.0% chr13 - 21893451 21893531 81 browser details YourSeq 36 1044 1105 3000 83.0% chr10 - 99094920 99094978 59
Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Tnfaip1 tumor necrosis factor, alpha-induced protein 1 (endothelial) [ Mus musculus (house mouse) ] Gene ID: 21927, updated on 10-Oct-2019
Gene summary
Official Symbol Tnfaip1 provided by MGI Official Full Name tumor necrosis factor, alpha-induced protein 1 (endothelial) provided by MGI Primary source MGI:MGI:104961 See related Ensembl:ENSMUSG00000017615 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 Edp1; Edp-1; Tnfip1; Bacurd2 Expression Ubiquitous expression in lung adult (RPKM 35.5), large intestine adult (RPKM 31.2) and 28 other tissues See more Orthologs human all
Genomic context
Location: 11 B5; 11 46.74 cM See Tnfaip1 in Genome Data Viewer
Exon count: 7
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (78522850..78536270, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (78336352..78349762, complement)
Chromosome 11 - NC_000077.6
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Transcript information: This gene has 2 transcripts
Gene: Tnfaip1 ENSMUSG00000017615
Description tumor necrosis factor, alpha-induced protein 1 (endothelial) [Source:MGI Symbol;Acc:MGI:104961] Gene Synonyms Bacurd2, Edp-1, Edp1, Tnfip1 Location Chromosome 11: 78,522,850-78,536,332 reverse strand. GRCm38:CM001004.2 About this gene This gene has 2 transcripts (splice variants), 193 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 24 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Tnfaip1-202 ENSMUST00000108277.2 3768 316aa ENSMUSP00000103912.2 Protein coding CCDS25110 O70479 TSL:1 GENCODE basic APPRIS P1
Tnfaip1-201 ENSMUST00000017759.8 3720 316aa ENSMUSP00000017759.2 Protein coding CCDS25110 O70479 TSL:1 GENCODE basic APPRIS P1
33.48 kb Forward strand 78.52Mb 78.53Mb 78.54Mb Genes Poldip2-201 >protein coding Ift20-203 >protein coding (Comprehensive set...
Poldip2-203 >lncRNA Poldip2-205 >retained intron Ift20-201 >protein coding
Poldip2-202 >nonsense mediated decay Ift20-202 >protein coding
Poldip2-204 >retained intron Ift20-204 >retained intron
Poldip2-206 >retained intron Ift20-205 >retained intron
Contigs AL591177.14 >
Genes (Comprehensive set... < Tnfaip1-202protein coding < Tmem97-201protein coding
< Tnfaip1-201protein coding
Regulatory Build
78.52Mb 78.53Mb 78.54Mb Reverse strand 33.48 kb
Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site
Gene Legend Protein Coding
merged Ensembl/Havana Ensembl protein coding
Non-Protein Coding
processed transcript RNA gene
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Transcript: ENSMUST00000108277
< Tnfaip1-202protein coding
Reverse strand 13.48 kb
ENSMUSP00000103... MobiDB lite Superfamily SKP1/BTB/POZ domain superfamily SMART BTB/POZ domain Pfam Potassium channel tetramerisation-type BTB domain PROSITE profiles BTB/POZ domain PANTHER PTHR11145:SF17
PTHR11145 Gene3D 3.30.710.10 CDD cd18401
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend synonymous variant
Scale bar 0 40 80 120 160 200 240 316
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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