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Mouse Tomm34 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Tomm34 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Tomm34 gene (NCBI Reference Sequence: NM_025996 ; Ensembl: ENSMUSG00000018322 ) is located on Mouse chromosome 2. 7 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000018466). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tomm34 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-399D16 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 null mice are fertile and males do not display any defects in the testes or in spermatogenesis.
Exon 2 starts from about 13.81% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 3797 bp, and the size of intron 2 for 3'-loxP site insertion: 1525 bp. The size of effective cKO region: ~600 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 2 3 7 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Tomm34 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(7100bp) | A(24.93% 1770) | C(22.17% 1574) | T(28.92% 2053) | G(23.99% 1703)
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% chr2 - 164066920 164069919 3000 browser details YourSeq 170 160 793 3000 85.3% chr3 + 101485441 101485854 414 browser details YourSeq 170 141 349 3000 93.0% chr18 + 61094574 61094797 224 browser details YourSeq 170 144 469 3000 85.8% chr1 + 128208877 128209102 226 browser details YourSeq 169 137 330 3000 92.6% chr13 + 55656648 55656838 191 browser details YourSeq 167 144 346 3000 89.7% chr8 - 70881689 70881886 198 browser details YourSeq 167 144 332 3000 92.5% chr15 + 83123344 83123529 186 browser details YourSeq 166 139 332 3000 94.2% chr11 + 120770365 120770558 194 browser details YourSeq 166 138 334 3000 93.2% chr11 + 82767924 82768136 213 browser details YourSeq 165 144 334 3000 93.1% chr7 + 27693692 27693881 190 browser details YourSeq 165 144 333 3000 92.5% chr10 + 113995080 113995267 188 browser details YourSeq 165 144 333 3000 92.5% chr10 + 81203250 81203437 188 browser details YourSeq 164 145 332 3000 92.3% chr12 - 102715839 102716021 183 browser details YourSeq 164 135 335 3000 92.8% chr10 - 127656560 127656765 206 browser details YourSeq 164 144 348 3000 88.0% chr11 + 120689911 120690109 199 browser details YourSeq 163 144 332 3000 93.0% chr19 - 57082039 57082226 188 browser details YourSeq 163 129 343 3000 86.3% chr1 - 135759237 135759443 207 browser details YourSeq 163 141 332 3000 92.8% chrX + 94024082 94024409 328 browser details YourSeq 163 146 332 3000 92.9% chr8 + 85486471 85486655 185 browser details YourSeq 163 145 330 3000 94.6% chr12 + 80930730 80930917 188
Note: The 3000 bp section upstream 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 3000 1 3000 3000 100.0% chr2 - 164063320 164066319 3000 browser details YourSeq 377 1526 2099 3000 93.2% chr1 - 124910341 124911047 707 browser details YourSeq 376 1521 2102 3000 89.6% chr17 + 46424124 46424609 486 browser details YourSeq 363 1514 2100 3000 89.6% chr3 - 55938012 55938520 509 browser details YourSeq 363 1521 2102 3000 88.9% chr15 - 6516761 6517226 466 browser details YourSeq 353 1523 2102 3000 89.9% chr3 + 135913065 135913590 526 browser details YourSeq 342 1527 2102 3000 91.0% chr3 - 65633136 65633692 557 browser details YourSeq 340 1541 2102 3000 91.7% chr12 - 105745601 105746356 756 browser details YourSeq 335 1480 2102 3000 94.7% chr4 - 72104539 72105170 632 browser details YourSeq 305 1778 2123 3000 96.1% chr4 + 133909239 133909796 558 browser details YourSeq 296 1779 2102 3000 95.7% chr7 + 130436474 130436797 324 browser details YourSeq 293 1691 2102 3000 94.9% chr9 - 104830163 104830738 576 browser details YourSeq 293 1780 2102 3000 95.7% chr4 + 37942629 37942952 324 browser details YourSeq 293 1776 2102 3000 95.1% chr1 + 16277519 16678774 401256 browser details YourSeq 292 1778 2100 3000 95.4% chrX + 15392614 15392937 324 browser details YourSeq 292 1779 2102 3000 95.4% chr11 + 77264195 77264519 325 browser details YourSeq 290 1778 2103 3000 94.5% chr7 - 64556843 64557168 326 browser details YourSeq 290 1779 2102 3000 95.1% chr4 - 32125826 32214772 88947 browser details YourSeq 289 1778 2102 3000 94.5% chr14 - 52552463 52552787 325 browser details YourSeq 288 1778 2103 3000 94.5% chrX - 16758993 16759319 327
Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Tomm34 translocase of outer mitochondrial membrane 34 [ Mus musculus (house mouse) ] Gene ID: 67145, updated on 10-Oct-2019
Gene summary
Official Symbol Tomm34 provided by MGI Official Full Name translocase of outer mitochondrial membrane 34 provided by MGI Primary source MGI:MGI:1914395 See related Ensembl:ENSMUSG00000018322 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 TOM34; 2610100K07Rik Expression Ubiquitous expression in adrenal adult (RPKM 25.7), thymus adult (RPKM 24.5) and 28 other tissues See more Orthologs human all
Genomic context
Location: 2; 2 H3 See Tomm34 in Genome Data Viewer
Exon count: 8
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (164053538..164071178, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (163879277..163896838, complement)
Chromosome 2 - NC_000068.7
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Transcript information: This gene has 3 transcripts
Gene: Tomm34 ENSMUSG00000018322
Description translocase of outer mitochondrial membrane 34 [Source:MGI Symbol;Acc:MGI:1914395] Gene Synonyms 2610100K07Rik, TOM34 Location Chromosome 2: 164,053,540-164,071,169 reverse strand. GRCm38:CM000995.2 About this gene This gene has 3 transcripts (splice variants), 125 orthologues, 18 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Tomm34-201 ENSMUST00000018466.3 2302 309aa ENSMUSP00000018466.3 Protein coding CCDS17020 Q9CYG7 TSL:1 GENCODE basic APPRIS P3
Tomm34-202 ENSMUST00000109384.9 1923 309aa ENSMUSP00000105010.3 Protein coding CCDS71187 Q9CYG7 TSL:1 GENCODE basic APPRIS ALT1
Tomm34-203 ENSMUST00000132602.1 2089 No protein - Retained intron - - TSL:1
37.63 kb Forward strand 164.05Mb 164.06Mb 164.07Mb 164.08Mb Genes Pabpc1l-201 >protein coding Stk4-203 >protein coding (Comprehensive set...
Pabpc1l-202 >lncRNA Stk4-201 >protein coding
Pabpc1l-204 >lncRNA
Contigs AL591542.20 > AL591512.11 > Genes (Comprehensive set... < Tomm34-203retained intron
< Tomm34-201protein coding
< Tomm34-202protein coding
Regulatory Build
164.05Mb 164.06Mb 164.07Mb 164.08Mb Reverse strand 37.63 kb
Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
merged Ensembl/Havana Ensembl protein coding
Non-Protein Coding
processed transcript RNA gene
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Transcript: ENSMUST00000018466
< Tomm34-201protein coding
Reverse strand 17.56 kb
ENSMUSP00000018... MobiDB lite Low complexity (Seg) Superfamily Tetratricopeptide-like helical domain superfamily SMART Tetratricopeptide repeat Pfam Tetratricopeptide repeat 1
Tetratricopeptide repeat PROSITE profiles Tetratricopeptide repeat
Tetratricopeptide repeat-containing domain PANTHER PTHR45984:SF2
PTHR45984 Gene3D Tetratricopeptide-like helical domain superfamily
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend
missense variant synonymous variant
Scale bar 0 40 80 120 160 200 240 309
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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