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Mouse Tmem8b Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tmem8b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tmem8b gene (NCBI Reference Sequence: NM_001085508 ; Ensembl: ENSMUSG00000078716 ) is located on Mouse chromosome 4. 13 exons are identified, with the ATG start codon in exon 6 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000107865). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tmem8b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-191F22 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 6 starts from about 100% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 644 bp, and the size of intron 6 for 3'-loxP site insertion: 3012 bp. The size of effective cKO region: ~826 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 4 5 6 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tmem8b 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(7279bp) | A(22.26% 1620) | C(25.11% 1828) | T(28.11% 2046) | G(24.52% 1785)

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 + 43678757 43681756 3000 browser details YourSeq 41 1175 1407 3000 95.6% chr17 + 35230081 35230313 233 browser details YourSeq 40 1249 1404 3000 62.1% chr2 - 5496206 5496274 69 browser details YourSeq 39 1310 1407 3000 88.1% chr9 + 105578439 105578534 96 browser details YourSeq 37 1328 1467 3000 93.1% chr11 + 61784333 61784476 144 browser details YourSeq 36 1285 1407 3000 62.5% chr15 - 12398679 12398769 91 browser details YourSeq 34 1388 1529 3000 94.8% chr10 + 60735538 60735681 144 browser details YourSeq 32 1358 1404 3000 85.0% chr3 - 9129161 9129206 46 browser details YourSeq 28 1309 1343 3000 93.8% chr10 + 115559541 115559576 36 browser details YourSeq 27 1656 1684 3000 96.6% chr14 - 45574016 45574044 29 browser details YourSeq 27 1391 1467 3000 67.6% chr1 - 181322850 181322926 77 browser details YourSeq 25 1310 1335 3000 100.0% chr6 + 142837969 142837996 28 browser details YourSeq 25 1310 1335 3000 100.0% chr12 + 102464089 102464116 28 browser details YourSeq 23 1310 1335 3000 96.2% chr18 - 34501451 34501478 28

Note: The 3000 bp section upstream of Exon 6 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 + 43682536 43685535 3000 browser details YourSeq 248 539 1325 3000 83.9% chr3 + 145489260 145489900 641 browser details YourSeq 236 540 878 3000 89.2% chr5 + 65406922 65407279 358 browser details YourSeq 233 535 878 3000 90.6% chr6 - 149212778 149213136 359 browser details YourSeq 229 550 871 3000 91.1% chrX - 105890061 105890424 364 browser details YourSeq 227 565 1325 3000 83.2% chr17 - 55920188 55920617 430 browser details YourSeq 226 540 1325 3000 82.7% chr7 - 101928970 101929433 464 browser details YourSeq 225 540 869 3000 90.0% chr12 + 71108030 71108806 777 browser details YourSeq 224 540 869 3000 86.1% chr17 + 78989425 78989751 327 browser details YourSeq 223 556 866 3000 87.7% chr17 - 29387624 29387955 332 browser details YourSeq 222 540 869 3000 89.1% chr12 - 69136948 69137472 525 browser details YourSeq 222 539 898 3000 88.7% chr4 + 42693878 42694259 382 browser details YourSeq 221 566 894 3000 87.2% chr12 - 75794172 75794493 322 browser details YourSeq 221 540 869 3000 89.7% chr8 + 46267849 46268441 593 browser details YourSeq 221 540 866 3000 89.0% chr13 + 13419292 13419628 337 browser details YourSeq 219 570 880 3000 89.0% chr11 - 84461836 84462167 332 browser details YourSeq 219 541 878 3000 87.9% chrX + 77237507 77237843 337 browser details YourSeq 218 570 872 3000 85.2% chr2 - 127939835 127940113 279 browser details YourSeq 218 541 870 3000 86.2% chr17 + 33896574 33896901 328 browser details YourSeq 217 540 877 3000 86.6% chr14 - 54811994 54812311 318

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tmem8b transmembrane protein 8B [ Mus musculus (house mouse) ] Gene ID: 242409, updated on 24-Oct-2019

Gene summary

Official Symbol Tmem8b provided by MGI Official Full Name transmembrane protein 8B provided by MGI Primary source MGI:MGI:2441680 See related Ensembl:ENSMUSG00000078716 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 4930500O05Rik Expression Broad expression in testis adult (RPKM 21.3), whole brain E14.5 (RPKM 8.4) and 22 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 A5-B1 See Tmem8b in Genome Data Viewer

Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (43668971..43692668)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (43681843..43705540)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 9 transcripts

Gene: Tmem8b ENSMUSG00000078716

Description transmembrane protein 8B [Source:MGI Symbol;Acc:MGI:2441680] Gene Synonyms 4930500O05Rik Location Chromosome 4: 43,668,971-43,692,668 forward strand. GRCm38:CM000997.2 About this gene This gene has 9 transcripts (splice variants), 194 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tmem8b-202 ENSMUST00000107865.8 4775 472aa ENSMUSP00000103497.2 Protein coding CCDS51168 B1AWJ5 TSL:1 GENCODE basic APPRIS P1

Tmem8b-201 ENSMUST00000107864.7 4726 472aa ENSMUSP00000103496.1 Protein coding CCDS51168 B1AWJ5 TSL:5 GENCODE basic APPRIS P1

Tmem8b-209 ENSMUST00000167153.7 4101 472aa ENSMUSP00000129760.1 Protein coding CCDS51168 B1AWJ5 TSL:1 GENCODE basic APPRIS P1

Tmem8b-203 ENSMUST00000107866.8 5102 931aa ENSMUSP00000103498.2 Protein coding - B1AWJ4 TSL:5 GENCODE basic

Tmem8b-206 ENSMUST00000143339.7 824 154aa ENSMUSP00000130133.1 Protein coding - E9Q7V8 CDS 3' incomplete TSL:5

Tmem8b-204 ENSMUST00000134869.7 1243 No protein - lncRNA - - TSL:5

Tmem8b-205 ENSMUST00000141864.1 741 No protein - lncRNA - - TSL:5

Tmem8b-208 ENSMUST00000154112.1 584 No protein - lncRNA - - TSL:3

Tmem8b-207 ENSMUST00000143774.1 521 No protein - lncRNA - - TSL:5

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43.70 kb Forward strand 43.66Mb 43.67Mb 43.68Mb 43.69Mb 43.70Mb Genes (Comprehensive set... Gm12481-201 >processed pseudogene Tmem8b-205 >lncRNA Tmem8b-208 >lncRNA

Tmem8b-202 >protein coding Gm23257-201 >rRNA

Tmem8b-209 >protein coding

Tmem8b-201 >protein coding

Tmem8b-203 >protein coding

Tmem8b-204 >lncRNA

Tmem8b-206 >protein coding

Tmem8b-207 >lncRNA

Contigs AL732626.8 > Genes < Fam221b-201protein coding < Olfr70-201protein coding (Comprehensive set...

< Fam221b-202retained intron

Regulatory Build

43.66Mb 43.67Mb 43.68Mb 43.69Mb 43.70Mb Reverse strand 43.70 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

pseudogene processed transcript RNA gene

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Transcript: ENSMUST00000107865

23.70 kb Forward strand

Tmem8b-202 >protein coding

ENSMUSP00000103... Transmembrane heli... Low complexity (Seg) Pfam NGX6/PGAP6/MYMK

PROSITE patterns EGF-like, conserved site

EGF-like, conserved site PANTHER PTHR14319:SF6

NGX6/PGAP6/MYMK

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

Variant Legend

missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 472

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

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