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Mouse Tmem43 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tmem43 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tmem43 (NCBI Reference Sequence: NM_028766 ; Ensembl: ENSMUSG00000030095 ) is located on Mouse 6. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000032183). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tmem43 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-53E13 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: In a high-throughput screen, female homozygous mutant mice exhibited an increased anxiety-like response during open field activity testing when compared with their gender-matched wild-type littermates and the historical mean. Homozygous KO or certain codon substitution mutants don't affect heart function.

Exon 2 starts from about 1.08% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 3346 bp, and the size of intron 4 for 3'-loxP site insertion: 772 bp. The size of effective cKO region: ~2056 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 12 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tmem43 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(8556bp) | A(23.31% 1994) | C(25.58% 2189) | T(24.26% 2076) | G(26.85% 2297)

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% chr6 + 91473996 91476995 3000 browser details YourSeq 32 826 969 3000 97.2% chr5 - 121378425 121378570 146 browser details YourSeq 22 1679 1700 3000 100.0% chr3 + 36694660 36694681 22 browser details YourSeq 21 2786 2806 3000 100.0% chr8 + 89011976 89011996 21

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% chr6 + 91479052 91482051 3000 browser details YourSeq 61 2475 2704 3000 80.2% chr2 + 155987671 155987927 257 browser details YourSeq 51 2483 2626 3000 86.8% chr10 + 127112218 127112448 231 browser details YourSeq 45 2641 2720 3000 76.8% chr2 + 31330544 31330621 78 browser details YourSeq 45 2641 2745 3000 79.3% chr11 + 28847031 28847168 138 browser details YourSeq 43 2475 2544 3000 83.1% chr19 + 46748829 46748899 71 browser details YourSeq 42 2475 2656 3000 62.3% chr4 + 103165360 103165424 65 browser details YourSeq 41 2641 2705 3000 81.6% chr15 - 31377282 31377346 65 browser details YourSeq 38 2637 2705 3000 97.6% chr5 - 134178127 134178195 69 browser details YourSeq 37 2489 2540 3000 86.3% chr5 - 134707376 134707427 52 browser details YourSeq 37 2638 2702 3000 78.5% chr5 - 122063375 122063439 65 browser details YourSeq 37 2468 2536 3000 93.2% chr4 - 132501016 132501090 75 browser details YourSeq 37 2508 2704 3000 95.2% chr7 + 101332010 101332324 315 browser details YourSeq 35 2613 2665 3000 94.9% chr11 - 52228024 52228076 53 browser details YourSeq 35 2611 2705 3000 87.9% chr2 + 118705145 118705238 94 browser details YourSeq 35 2479 2530 3000 84.7% chr10 + 37296621 37296673 53 browser details YourSeq 34 2475 2657 3000 57.5% chr2 - 18070723 18070787 65 browser details YourSeq 34 2642 2705 3000 76.6% chr15 + 99956466 99956529 64 browser details YourSeq 33 2648 2705 3000 83.8% chr5 - 122335280 122335336 57 browser details YourSeq 33 2641 2705 3000 75.4% chr4 - 8571040 8571104 65

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Tmem43 transmembrane protein 43 [ Mus musculus (house mouse) ] Gene ID: 74122, updated on 10-Sep-2019

Gene summary

Official Symbol Tmem43 provided by MGI Official Full Name transmembrane protein 43 provided by MGI Primary source MGI:MGI:1921372 See related Ensembl:ENSMUSG00000030095 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 LUMA; 1200015A22Rik Expression Ubiquitous expression in subcutaneous fat pad adult (RPKM 68.5), mammary gland adult (RPKM 49.0) and 26 other Orthologs tissues See more human all

Genomic context

Location: 6; 6 D1 See Tmem43 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (91473707..91488463)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (91423745..91438452)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 5 transcripts

Gene: Tmem43 ENSMUSG00000030095

Description transmembrane protein 43 [Source:MGI Symbol;Acc:MGI:1921372] Gene Synonyms 1200015A22Rik, LUMA Location Chromosome 6: 91,473,703-91,488,463 forward strand. GRCm38:CM000999.2 About this gene This gene has 5 transcripts (splice variants), 195 orthologues, 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

Tmem43-201 ENSMUST00000032183.5 2903 400aa ENSMUSP00000032183.4 Protein coding CCDS20368 Q9DBS1 TSL:1 GENCODE basic APPRIS P1

Tmem43-202 ENSMUST00000140246.7 860 No protein - Retained intron - - TSL:2

Tmem43-204 ENSMUST00000153179.1 561 No protein - Retained intron - - TSL:2

Tmem43-203 ENSMUST00000144246.1 465 No protein - lncRNA - - TSL:3

Tmem43-205 ENSMUST00000205954.1 376 No protein - lncRNA - - TSL:5

34.76 kb Forward strand

91.47Mb 91.48Mb 91.49Mb (Comprehensive set... Tmem43-201 >protein coding

Tmem43-204 >retained intron

Tmem43-205 >lncRNA

Tmem43-202 >retained intron

Tmem43-203 >lncRNA

Contigs < AC161456.3 Genes < Chchd4-201protein coding < Xpc-201protein coding (Comprehensive set...

< Chchd4-202retained intron < Xpc-202nonsense mediated decay

Regulatory Build

91.47Mb 91.48Mb 91.49Mb Reverse strand 34.76 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000032183

14.76 kb Forward strand

Tmem43-201 >protein coding

ENSMUSP00000032... Transmembrane heli... Pfam Transmembrane protein 43 family

PANTHER PTHR13416:SF2

Transmembrane protein 43 family

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

Variant Legend

synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400

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

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