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Mouse Tmx4 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tmx4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tmx4 gene (NCBI Reference Sequence: NM_029148 ; Ensembl: ENSMUSG00000034723 ) is located on Mouse chromosome 2. 8 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000038228). 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 Tmx4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-20P22 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 2 starts from about 16.42% 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: 4004 bp, and the size of intron 2 for 3'-loxP site insertion: 18288 bp. The size of effective cKO region: ~834 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 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tmx4 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(7116bp) | A(31.1% 2213) | C(16.36% 1164) | T(34.43% 2450) | G(18.11% 1289)

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 - 134640088 134643087 3000 browser details YourSeq 91 1122 1664 3000 93.6% chr3 - 68569639 68570254 616 browser details YourSeq 84 1505 1667 3000 87.2% chr1 + 185740651 185740809 159 browser details YourSeq 82 1313 1668 3000 92.0% chr11 + 109128920 109129523 604 browser details YourSeq 81 1308 1667 3000 92.8% chr1 - 76008683 76009071 389 browser details YourSeq 79 1540 1665 3000 95.5% chr10 - 126553454 126553593 140 browser details YourSeq 77 1519 1656 3000 82.3% chr4 - 120328405 120328524 120 browser details YourSeq 74 1500 1658 3000 83.2% chr12 - 85740861 85740993 133 browser details YourSeq 71 1566 1674 3000 80.8% chr12 + 23980092 23980172 81 browser details YourSeq 69 1501 1614 3000 89.8% chr5 - 33454150 33454261 112 browser details YourSeq 66 1514 1632 3000 88.6% chr11 + 90325674 90325977 304 browser details YourSeq 65 1309 1658 3000 92.2% chr1 + 39507246 39507602 357 browser details YourSeq 64 1555 1654 3000 95.9% chr19 - 10348924 10349159 236 browser details YourSeq 63 1316 1577 3000 93.3% chr11 + 114219482 114219823 342 browser details YourSeq 59 1556 1663 3000 80.0% chr10 + 68860316 68860411 96 browser details YourSeq 58 1563 1667 3000 77.8% chr11 - 44981193 44981263 71 browser details YourSeq 57 1605 1667 3000 96.8% chr9 - 42408222 42408330 109 browser details YourSeq 56 1554 1658 3000 93.8% chr11 - 89343139 89343419 281 browser details YourSeq 55 1510 1667 3000 76.1% chr4 - 137065387 137065526 140 browser details YourSeq 55 1601 1666 3000 95.4% chr10 + 3865777 3865920 144

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 - 134636472 134639471 3000 browser details YourSeq 87 1255 1386 3000 84.7% chr12 + 83517302 83834723 317422 browser details YourSeq 79 1294 1420 3000 91.8% chr12 + 110973759 110973885 127 browser details YourSeq 77 1255 1419 3000 86.6% chr15 - 79204307 79204472 166 browser details YourSeq 77 1212 1414 3000 82.3% chr3 + 34760673 34760875 203 browser details YourSeq 73 1208 1420 3000 82.2% chr15 - 71912150 71912345 196 browser details YourSeq 73 1263 1420 3000 84.8% chr5 + 151087533 151087724 192 browser details YourSeq 72 1270 1416 3000 89.3% chr4 - 32910697 32910850 154 browser details YourSeq 69 1272 1420 3000 82.9% chr6 - 73271945 73272095 151 browser details YourSeq 66 1286 1420 3000 89.3% chr8 - 105809657 105809792 136 browser details YourSeq 66 2683 2771 3000 90.0% chr17 - 57884136 57884220 85 browser details YourSeq 65 2573 2894 3000 72.6% chrX + 111730134 111730396 263 browser details YourSeq 64 2576 2894 3000 70.0% chr3 - 69822037 69822236 200 browser details YourSeq 64 1270 1478 3000 94.6% chr4 + 134205832 134206316 485 browser details YourSeq 64 1331 1420 3000 85.6% chr14 + 52283429 52283518 90 browser details YourSeq 64 1265 1569 3000 92.4% chr11 + 61035685 61036050 366 browser details YourSeq 63 1264 1420 3000 82.3% chr4 - 138294661 138294817 157 browser details YourSeq 62 1272 1420 3000 88.8% chr1 - 188313821 188313970 150 browser details YourSeq 61 1270 1420 3000 85.1% chr7 + 30338490 30338641 152 browser details YourSeq 61 1347 1551 3000 82.8% chr7 + 16982121 16982679 559

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: Tmx4 thioredoxin-related transmembrane protein 4 [ Mus musculus (house mouse) ] Gene ID: 52837, updated on 10-Oct-2019

Gene summary

Official Symbol Tmx4 provided by MGI Official Full Name thioredoxin-related transmembrane protein 4 provided by MGI Primary source MGI:MGI:106558 See related Ensembl:ENSMUSG00000034723 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Txndc13; AI843224; AW046784; mKIAA1162; D2Bwg1356e; 2810417D04Rik; 4930500L08Rik Expression Broad expression in frontal lobe adult (RPKM 23.2), cerebellum adult (RPKM 18.6) and 21 other tissues See more Orthologs human all

Genomic context

Location: 2 F2; 2 65.66 cM See Tmx4 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (134594501..134644121, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (134420237..134469857, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 4 transcripts

Gene: Tmx4 ENSMUSG00000034723

Description thioredoxin-related transmembrane protein 4 [Source:MGI Symbol;Acc:MGI:106558] Gene Synonyms 2810417D04Rik, 4930500L08Rik, D2Bwg1356e, Txndc13 Location Chromosome 2: 134,594,185-134,644,145 reverse strand. GRCm38:CM000995.2 About this gene This gene has 4 transcripts (splice variants), 148 orthologues, 13 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tmx4-201 ENSMUST00000038228.10 5488 335aa ENSMUSP00000045154.4 Protein coding CCDS16784 Q0P5W2 Q8C0L0 TSL:1 GENCODE basic APPRIS P2

Tmx4-203 ENSMUST00000110120.1 2602 183aa ENSMUSP00000105747.1 Protein coding - A2ARI0 TSL:1 GENCODE basic APPRIS ALT2

Tmx4-202 ENSMUST00000110119.1 596 166aa ENSMUSP00000105746.1 Protein coding - A2ARI1 TSL:2 GENCODE basic

Tmx4-204 ENSMUST00000137377.1 3917 No protein - lncRNA - - TSL:1

69.96 kb Forward strand 134.60Mb 134.62Mb 134.64Mb Contigs AL845418.17 > Genes (Comprehensive set... < Tmx4-201protein coding

< Tmx4-204lncRNA

< Tmx4-203protein coding

< Tmx4-202protein coding

Regulatory Build

134.60Mb 134.62Mb 134.64Mb Reverse strand 69.96 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000038228

< Tmx4-201protein coding

Reverse strand 49.96 kb

ENSMUSP00000045... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Cleavage site (Sign... Superfamily Thioredoxin-like superfamily Pfam Thioredoxin domain PROSITE profiles Thioredoxin domain PROSITE patterns Thioredoxin, conserved site PANTHER PTHR46107

PTHR46107:SF1 Gene3D 3.40.30.10

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 335

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

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