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Mouse Tmem131 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tmem131 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tmem131 gene (NCBI Reference Sequence: NM_018872 ; Ensembl: ENSMUSG00000026116 ) is located on Mouse chromosome 1. 41 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 41 (Transcript: ENSMUST00000027290). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tmem131 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-75F3 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 5 starts from about 6.29% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 17544 bp, and the size of intron 5 for 3'-loxP site insertion: 13133 bp. The size of effective cKO region: ~624 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 5 41 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tmem131 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(7124bp) | A(26.95% 1920) | C(20.62% 1469) | T(29.9% 2130) | G(22.53% 1605)

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% chr1 - 36855218 36858217 3000 browser details YourSeq 48 122 209 3000 77.3% chr2 + 30698401 30698488 88 browser details YourSeq 43 136 209 3000 86.5% chr7 - 135797298 135797372 75 browser details YourSeq 39 135 211 3000 75.4% chrX - 52223965 52224041 77 browser details YourSeq 39 101 182 3000 93.2% chr1 - 128081538 128081621 84 browser details YourSeq 39 135 213 3000 74.7% chr2 + 167095335 167095413 79 browser details YourSeq 37 93 153 3000 93.2% chr1 + 56880256 56880316 61 browser details YourSeq 35 1536 1746 3000 92.7% chr14 - 90204016 90204226 211 browser details YourSeq 35 139 209 3000 85.0% chr1 - 106520743 106520811 69 browser details YourSeq 33 88 151 3000 91.5% chr17 - 84869541 84869603 63 browser details YourSeq 33 135 209 3000 72.0% chr1 - 150508578 150508652 75 browser details YourSeq 30 135 180 3000 82.7% chr12 - 59019656 59019701 46 browser details YourSeq 30 163 213 3000 94.2% chr11 + 86115393 86115444 52 browser details YourSeq 30 103 208 3000 64.2% chr1 + 89396155 89396260 106 browser details YourSeq 29 96 150 3000 87.9% chr19 + 21315234 21315287 54 browser details YourSeq 28 1859 1889 3000 86.3% chr1 - 143705066 143705094 29 browser details YourSeq 27 135 182 3000 89.7% chr5 - 72260240 72260286 47 browser details YourSeq 27 1500 1547 3000 89.7% chr1 - 144337927 144337973 47 browser details YourSeq 27 89 115 3000 100.0% chr3 + 141376555 141376581 27 browser details YourSeq 26 1470 1510 3000 89.3% chr15 - 66090824 66090863 40

Note: The 3000 bp section upstream of Exon 5 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% chr1 - 36851594 36854593 3000 browser details YourSeq 200 1178 1430 3000 92.7% chr14 - 52079138 52079342 205 browser details YourSeq 200 1186 1430 3000 96.3% chr12 + 69176981 69177363 383 browser details YourSeq 194 1241 1622 3000 97.6% chr4 + 156246214 156246624 411 browser details YourSeq 193 1218 1430 3000 99.0% chr10 - 77526283 77526531 249 browser details YourSeq 192 1241 1436 3000 99.0% chr14 - 54600066 54600261 196 browser details YourSeq 191 1242 1446 3000 98.5% chrX + 60911433 60911641 209 browser details YourSeq 190 1240 1431 3000 99.5% chr8 - 34822323 34822514 192 browser details YourSeq 190 1238 1430 3000 99.5% chr3 + 152545803 152546000 198 browser details YourSeq 190 1241 1430 3000 100.0% chr1 + 133553492 133553681 190 browser details YourSeq 189 1222 1431 3000 94.0% chrY - 17001713 17001910 198 browser details YourSeq 189 1241 1433 3000 99.0% chr8 - 88307269 88307461 193 browser details YourSeq 189 1219 1430 3000 93.8% chr4 - 116197283 116197475 193 browser details YourSeq 189 1239 1430 3000 99.5% chr4 - 99099376 99099569 194 browser details YourSeq 189 1241 1430 3000 100.0% chr17 - 37980258 37980448 191 browser details YourSeq 189 1241 1431 3000 99.5% chr8 + 111810223 111810413 191 browser details YourSeq 189 1243 1431 3000 100.0% chr6 + 115731999 115732187 189 browser details YourSeq 189 1241 1435 3000 97.5% chr18 + 59083994 59084187 194 browser details YourSeq 188 1241 1430 3000 99.5% chrX - 71160530 71160719 190 browser details YourSeq 188 1241 1432 3000 99.0% chr14 - 54831598 54831789 192

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tmem131 transmembrane protein 131 [ Mus musculus (house mouse) ] Gene ID: 56030, updated on 12-Aug-2019

Gene summary

Official Symbol Tmem131 provided by MGI Official Full Name transmembrane protein 131 provided by MGI Primary source MGI:MGI:1927110 See related Ensembl:ENSMUSG00000026116 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 Neg; RW1; CC28; YR-23; mKIAA0257; D1Bwg0491e; 2610524E03Rik Expression Ubiquitous expression in thymus adult (RPKM 16.9), colon adult (RPKM 15.5) and 28 other tissues See more Orthologs human all

Genomic context

Location: 1 B; 1 15.42 cM See Tmem131 in Genome Data Viewer

Exon count: 42

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (36792189..36939614, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (36849034..36996372, complement)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 8 transcripts

Gene: Tmem131 ENSMUSG00000026116

Description transmembrane protein 131 [Source:MGI Symbol;Acc:MGI:1927110] Gene Synonyms 2610524E03Rik, CC28, D1Bwg0491e, Neg, Rw1, YR-23 Location Chromosome 1: 36,792,191-36,943,666 reverse strand. GRCm38:CM000994.2 About this gene This gene has 8 transcripts (splice variants), 216 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tmem131- ENSMUST00000194563.5 6698 1877aa ENSMUSP00000142307.1 Protein coding CCDS35538 O70472 TSL:5 208 GENCODE basic APPRIS P1

Tmem131- ENSMUST00000027290.11 6546 1877aa ENSMUSP00000027290.5 Protein coding CCDS35538 O70472 TSL:1 201 GENCODE basic APPRIS P1

Tmem131- ENSMUST00000189470.1 696 146aa ENSMUSP00000140620.1 Protein coding - A0A087WRG7 CDS 5' 205 incomplete TSL:3

Tmem131- ENSMUST00000185964.2 591 61aa ENSMUSP00000141413.1 Protein coding - A0A0A6YW65 CDS 3' 202 incomplete TSL:3

Tmem131- ENSMUST00000190442.1 515 171aa ENSMUSP00000140187.1 Protein coding - A0A087WQG5 CDS 5' and 3' 206 incomplete TSL:3

Tmem131- ENSMUST00000186486.1 682 56aa ENSMUSP00000142080.1 Nonsense mediated - A0A0A6YXP8 CDS 5' 203 decay incomplete TSL:3

Tmem131- ENSMUST00000191381.1 3969 No - Retained intron - - TSL:1 207 protein

Tmem131- ENSMUST00000187917.1 703 No - Retained intron - - TSL:3 204 protein

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171.48 kb Forward strand

36.80Mb 36.85Mb 36.90Mb 36.95Mb Genes Zap70-201 >protein coding (Comprehensive set...

Zap70-204 >retained intron

Contigs < AC084389.1 < AC123854.3 Genes (Comprehensive set... < Tmem131-201protein coding

< Tmem131-208protein coding

< Tmem131-207retained intron < Tmem131-202protein coding

< Tmem131-204retained intron < Gm37506-201TEC < Gm38115-201lncRNA

< Tmem131-205protein coding < F830112A20Rik-201TEC

< Tmem131-206protein coding

< Tmem131-203nonsense mediated decay

Regulatory Build

36.80Mb 36.85Mb 36.90Mb 36.95Mb Reverse strand 171.48 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000027290

< Tmem131-201protein coding

Reverse strand 147.34 kb

protein_pic

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

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