https://www.alphaknockout.com Mouse Tinagl1 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Tinagl1 conditional knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Tinagl1 gene (NCBI Reference Sequence: NM_023476 ; Ensembl: ENSMUSG00000028776 ) is located on Mouse chromosome 4. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000105998). 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 Tinagl1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-282L14 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: Female mice homozygous for a null mutation display impaired fertility and homozygous pups born to homozygous females show impaired postnatal survival.
Exon 4 starts from about 26.61% 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: 3528 bp, and the size of intron 5 for 3'-loxP site insertion: 965 bp. The size of effective cKO region: ~885 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 7 8 9 10 12 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Tinagl1 Homology arm cKO region loxP site
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Overview of the Dot Plot Window size: 10 bp
Forward Reverse Complement
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
Summary: Full Length(7385bp) | A(22.76% 1681) | C(25.59% 1890) | T(23.1% 1706) | G(28.54% 2108)
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% chr4 - 130169654 130172653 3000 browser details YourSeq 167 1703 1884 3000 96.7% chrX + 74277818 74278006 189 browser details YourSeq 159 1703 1882 3000 94.9% chrX + 143117796 143117977 182 browser details YourSeq 152 1703 1882 3000 91.1% chr8 - 89122891 89123060 170 browser details YourSeq 152 1706 1878 3000 92.0% chr18 - 38353884 38354044 161 browser details YourSeq 152 1710 1878 3000 95.9% chr11 - 76316649 76317215 567 browser details YourSeq 148 1705 1878 3000 90.9% chr11 + 66659610 66659775 166 browser details YourSeq 147 1701 1875 3000 90.7% chr11 + 31943469 31943630 162 browser details YourSeq 146 1701 1882 3000 90.8% chr3 - 70618822 70619001 180 browser details YourSeq 146 1703 1877 3000 92.5% chr10 - 87445496 87445670 175 browser details YourSeq 142 1734 1886 3000 96.8% chr11 - 86785198 86785352 155 browser details YourSeq 142 1714 1882 3000 92.0% chr4 + 82371965 82372131 167 browser details YourSeq 142 1703 1879 3000 88.5% chr3 + 9008138 9008303 166 browser details YourSeq 141 1734 1878 3000 98.7% chr17 + 27900487 27900631 145 browser details YourSeq 139 1714 1878 3000 89.4% chr4 + 24371072 24371230 159 browser details YourSeq 138 1734 1878 3000 98.0% chr6 - 31430883 31431030 148 browser details YourSeq 138 1702 1875 3000 89.8% chr5 - 96920182 96920346 165 browser details YourSeq 138 1714 1878 3000 91.9% chr2 - 26682406 26682569 164 browser details YourSeq 138 1726 1878 3000 96.0% chr7 + 141421060 141421214 155 browser details YourSeq 137 1734 1877 3000 98.0% chr9 - 101032586 101032731 146
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% chr4 - 130165769 130168768 3000 browser details YourSeq 41 2138 2243 3000 90.4% chr2 + 3290866 3290978 113 browser details YourSeq 38 2217 2256 3000 100.0% chr4 - 57834691 57834807 117 browser details YourSeq 28 2219 2255 3000 96.7% chr13 - 24739452 24739492 41 browser details YourSeq 26 1440 1465 3000 100.0% chr11 + 41165775 41165800 26 browser details YourSeq 25 2543 2570 3000 84.7% chr6 - 30605426 30605451 26 browser details YourSeq 24 2218 2243 3000 88.0% chr2 - 158090457 158090481 25 browser details YourSeq 24 2217 2242 3000 88.0% chr11 - 115058912 115058936 25 browser details YourSeq 24 2051 2074 3000 100.0% chr10 - 85064159 85064182 24 browser details YourSeq 24 743 772 3000 84.7% chr8 + 99206130 99206157 28 browser details YourSeq 24 2218 2243 3000 96.2% chr14 + 56461186 56461211 26 browser details YourSeq 24 2218 2243 3000 88.0% chr14 + 48597071 48597095 25 browser details YourSeq 23 271 293 3000 100.0% chr1 - 130898621 130898643 23 browser details YourSeq 22 2215 2236 3000 100.0% chr17 - 52124441 52124462 22 browser details YourSeq 22 2104 2125 3000 100.0% chr17 - 10310190 10310211 22 browser details YourSeq 22 2053 2074 3000 100.0% chr1 - 75329421 75329442 22 browser details YourSeq 22 1445 1468 3000 95.9% chr7 + 34915556 34915579 24 browser details YourSeq 22 259 280 3000 100.0% chr16 + 8113525 8113546 22 browser details YourSeq 22 549 570 3000 100.0% chr13 + 40521835 40521856 22 browser details YourSeq 21 148 168 3000 100.0% chr10 - 88642461 88642481 21
Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.
Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Tinagl1 tubulointerstitial nephritis antigen-like 1 [ Mus musculus (house mouse) ] Gene ID: 94242, updated on 12-Aug-2019
Gene summary
Official Symbol Tinagl1 provided by MGI Official Full Name tubulointerstitial nephritis antigen-like 1 provided by MGI Primary source MGI:MGI:2137617 See related Ensembl:ENSMUSG00000028776 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 AZ1; AZ-1; Arg1; Lcn7; TARP; Tinagl; 1110021J17Rik Expression Broad expression in bladder adult (RPKM 158.0), placenta adult (RPKM 140.0) and 19 other tissues See more Orthologs human all
Genomic context
Location: 4; 4 D2.2 See Tinagl1 in Genome Data Viewer Exon count: 13
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (130165600..130175122, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (129842844..129852366, complement)
Chromosome 4 - NC_000070.6
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Transcript information: This gene has 9 transcripts
Gene: Tinagl1 ENSMUSG00000028776
Description tubulointerstitial nephritis antigen-like 1 [Source:MGI Symbol;Acc:MGI:2137617] Gene Synonyms 1110021J17Rik, AZ-1, Arg1, Lcn7, androgen-regulated gene 1 Location Chromosome 4: 130,164,454-130,175,122 reverse strand. GRCm38:CM000997.2 About this gene This gene has 9 transcripts (splice variants), 198 orthologues, 27 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Tinagl1- ENSMUST00000105998.7 1976 466aa ENSMUSP00000101620.1 Protein coding CCDS18708 Q99JR5 TSL:1 202 GENCODE basic APPRIS P2
Tinagl1- ENSMUST00000105999.8 1952 466aa ENSMUSP00000101621.2 Protein coding CCDS18708 Q99JR5 TSL:1 203 GENCODE basic APPRIS P2
Tinagl1- ENSMUST00000175992.7 1847 435aa ENSMUSP00000134900.1 Protein coding - H3BJ97 TSL:5 209 GENCODE basic APPRIS ALT2
Tinagl1- ENSMUST00000132545.2 977 161aa ENSMUSP00000135453.1 Protein coding - H3BKM8 CDS 3' 204 incomplete TSL:2
Tinagl1- ENSMUST00000030560.8 2085 466aa ENSMUSP00000030560.2 Nonsense mediated - Q99JR5 TSL:1 201 decay
Tinagl1- ENSMUST00000145000.2 911 No - Retained intron - - TSL:2 206 protein
Tinagl1- ENSMUST00000145774.2 849 No - Retained intron - - TSL:5 207 protein
Tinagl1- ENSMUST00000133660.2 596 No - Retained intron - - TSL:1 205 protein
Tinagl1- ENSMUST00000175822.1 1100 No - lncRNA - - TSL:5 208 protein
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30.67 kb Forward strand 130.16Mb 130.17Mb 130.18Mb Contigs AL606925.16 >
Genes < Tinagl1-201nonsense mediated decay (Comprehensive set...
< Tinagl1-203protein coding
< Tinagl1-209protein coding
< Tinagl1-206retained intron < Tinagl1-208lncRNA
< Tinagl1-202protein coding
< Tinagl1-207retained intron < Tinagl1-205retained intron
< Tinagl1-204protein coding
Regulatory Build
130.16Mb 130.17Mb 130.18Mb Reverse strand 30.67 kb
Regulation Legend
CTCF Enhancer Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
Non-Protein Coding
RNA gene processed transcript
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Transcript: ENSMUST00000105998
< Tinagl1-202protein coding
Reverse strand 9.40 kb
ENSMUSP00000101... Low complexity (Seg) Cleavage site (Sign... Superfamily Papain-like cysteine peptidase superfamily SMART Peptidase C1A, papain C-terminal Pfam Peptidase C1A, papain C-terminal PROSITE profiles Somatomedin B domain PROSITE patterns Somatomedin B domain Cysteine peptidase, histidine active site
PANTHER PTHR12411:SF270
PTHR12411 Gene3D 3.90.70.10 CDD cd02620
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant splice region variant synonymous variant
Scale bar 0 40 80 120 160 200 240 280 320 360 400 466
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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