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Mouse Tpd52l2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tpd52l2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tpd52l2 gene (NCBI Reference Sequence: NM_025482 ; Ensembl: ENSMUSG00000000827 ) is located on Mouse chromosome 2. 8 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 8 (Transcript: ENSMUST00000149163). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tpd52l2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-60M5 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: Female mice homozygous for a knock-out allele exhibit decreased body length and absent or minimal hepatic lipidosis.

Exon 2 starts from about 3.03% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 2512 bp, and the size of intron 3 for 3'-loxP site insertion: 822 bp. The size of effective cKO region: ~2709 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 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tpd52l2 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(9209bp) | A(25.44% 2343) | C(21.61% 1990) | T(29.67% 2732) | G(23.28% 2144)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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 + 181496596 181499595 3000 browser details YourSeq 279 1592 2343 3000 84.6% chr5 + 67072985 67165266 92282 browser details YourSeq 239 1765 2296 3000 78.3% chr19 + 3689599 3690090 492 browser details YourSeq 235 1728 2332 3000 80.9% chr19 + 42480684 42481298 615 browser details YourSeq 220 1614 2296 3000 82.6% chr19 + 25578225 25578946 722 browser details YourSeq 213 1565 2331 3000 77.4% chr10 - 86424915 86425477 563 browser details YourSeq 201 1791 2291 3000 86.4% chr9 - 106365523 106583352 217830 browser details YourSeq 190 1566 2343 3000 75.5% chr13 + 47935463 47935966 504 browser details YourSeq 184 1832 2343 3000 74.1% chr16 - 89804042 89804516 475 browser details YourSeq 181 1600 1939 3000 94.6% chr11 - 90656109 90658934 2826 browser details YourSeq 179 1820 2367 3000 79.0% chr6 + 97606975 97607483 509 browser details YourSeq 176 1782 2735 3000 77.0% chr6 + 91968742 91969330 589 browser details YourSeq 176 1822 2351 3000 78.3% chr2 + 167809454 167809804 351 browser details YourSeq 174 1819 2354 3000 77.6% chr3 - 144624813 144625244 432 browser details YourSeq 167 1903 2349 3000 84.1% chr16 + 78294872 78295344 473 browser details YourSeq 165 1839 2328 3000 83.2% chr9 - 67812635 67813095 461 browser details YourSeq 164 1565 2343 3000 77.9% chr2 + 10194526 10195066 541 browser details YourSeq 163 1666 2287 3000 90.0% chrX - 13058317 13058937 621 browser details YourSeq 159 1807 2292 3000 78.1% chr9 + 84059963 84060412 450 browser details YourSeq 158 1533 2213 3000 82.4% chr6 - 51348825 51349468 644

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 + 181502305 181505304 3000 browser details YourSeq 205 1602 1998 3000 85.6% chr5 + 45652577 45652999 423 browser details YourSeq 180 1577 1922 3000 82.4% chr6 - 97139252 97139834 583 browser details YourSeq 167 1516 2000 3000 82.5% chr12 - 72412803 72413302 500 browser details YourSeq 167 940 1627 3000 95.7% chr11 + 98904181 99308571 404391 browser details YourSeq 166 1577 1994 3000 88.5% chr15 - 26461805 26462331 527 browser details YourSeq 163 927 1880 3000 94.1% chr18 + 39842428 40076872 234445 browser details YourSeq 162 1577 1995 3000 81.7% chr7 - 26682953 26683398 446 browser details YourSeq 158 1544 1922 3000 83.3% chr7 + 117707959 117708351 393 browser details YourSeq 153 1585 2024 3000 82.0% chr9 - 20486856 20487280 425 browser details YourSeq 150 912 1079 3000 96.4% chr11 - 33507714 33507889 176 browser details YourSeq 149 1580 1980 3000 88.5% chr14 - 56876480 56876903 424 browser details YourSeq 146 1763 1996 3000 87.7% chr11 - 56980858 56981093 236 browser details YourSeq 145 927 1080 3000 97.4% chr9 - 64224242 64224402 161 browser details YourSeq 145 927 1082 3000 96.8% chr11 - 70548655 70548813 159 browser details YourSeq 144 929 1091 3000 94.5% chr11 - 117835646 117836171 526 browser details YourSeq 142 926 1080 3000 96.2% chr16 - 35003183 35003339 157 browser details YourSeq 142 927 1079 3000 96.8% chr14 + 32112416 32112570 155 browser details YourSeq 141 1545 1919 3000 87.9% chr8 - 87548575 87548945 371 browser details YourSeq 141 1506 1909 3000 81.5% chr4 - 99103905 99104302 398

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tpd52l2 tumor protein D52-like 2 [ Mus musculus (house mouse) ] Gene ID: 66314, updated on 24-Oct-2019

Gene summary

Official Symbol Tpd52l2 provided by MGI Official Full Name tumor protein D52-like 2 provided by MGI Primary source MGI:MGI:1913564 See related Ensembl:ENSMUSG00000000827 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 D54; C86069; AU016537; 2810411G23Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 19.2), bladder adult (RPKM 18.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 H4 See Tpd52l2 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (181497142..181517962)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (181231958..181251462)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 12 transcripts

Gene: Tpd52l2 ENSMUSG00000000827

Description tumor protein D52-like 2 [Source:MGI Symbol;Acc:MGI:1913564] Gene Synonyms 2810411G23Rik, D54 Location Chromosome 2: 181,497,142-181,517,966 forward strand. GRCm38:CM000995.2 About this gene This gene has 12 transcripts (splice variants), 265 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tpd52l2- ENSMUST00000149163.7 3603 220aa ENSMUSP00000117690.1 Protein coding CCDS17214 Q9CYZ2 TSL:1 208 GENCODE basic

Tpd52l2- ENSMUST00000108800.7 3475 186aa ENSMUSP00000104428.1 Protein coding CCDS71221 Q3TUJ9 TSL:1 204 GENCODE basic APPRIS ALT1

Tpd52l2- ENSMUST00000000844.14 2950 206aa ENSMUSP00000000844.8 Protein coding CCDS71219 Q3TAI4 TSL:1 201 GENCODE basic

Tpd52l2- ENSMUST00000069712.8 1799 200aa ENSMUSP00000068888.2 Protein coding CCDS71220 Q8BKP1 TSL:1 202 GENCODE basic APPRIS P4

Tpd52l2- ENSMUST00000108799.9 985 229aa ENSMUSP00000104427.3 Protein coding - A2AUD5 TSL:5 203 GENCODE basic

Tpd52l2- ENSMUST00000184849.7 854 169aa ENSMUSP00000138837.1 Protein coding - V9GWU5 CDS 3' 212 incomplete TSL:3

Tpd52l2- ENSMUST00000141825.1 484 161aa ENSMUSP00000123627.1 Protein coding - F6VQ81 CDS 5' and 3' 207 incomplete TSL:5

Tpd52l2- ENSMUST00000184588.7 587 45aa ENSMUSP00000139238.1 Nonsense mediated - V9GXN2 TSL:3 211 decay

Tpd52l2- ENSMUST00000129390.1 900 No - Retained intron - - TSL:2 205 protein

Tpd52l2- ENSMUST00000130261.1 764 No - Retained intron - - TSL:2 206 protein

Tpd52l2- ENSMUST00000155589.1 549 No - Retained intron - - TSL:3 209 protein

Tpd52l2- ENSMUST00000183956.7 620 No - lncRNA - - TSL:3 210 protein

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40.83 kb Forward strand 181.49Mb 181.50Mb 181.51Mb 181.52Mb Genes Abhd16b-201 >protein codingTpd52l2-207 >protein coding Dnajc5-204 >nonsense mediated decay (Comprehensive set...

Tpd52l2-211 >nonsense mediated decay Dnajc5-201 >protein coding

Tpd52l2-210 >lncRNA Gm25184-201 >snRNA Dnajc5-203 >protein coding

Tpd52l2-208 >protein coding Dnajc5-206 >protein coding

Tpd52l2-201 >protein coding Dnajc5-202 >protein coding

Tpd52l2-212 >protein coding

Tpd52l2-204 >protein coding

Tpd52l2-202 >protein coding

Tpd52l2-205 >retained intron Tpd52l2-206 >retained intron

Tpd52l2-203 >protein coding

Tpd52l2-209 >retained intron

Contigs AL929094.13 > Regulatory Build

181.49Mb 181.50Mb 181.51Mb 181.52Mb Reverse strand 40.83 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000149163

20.80 kb Forward strand

Tpd52l2-208 >protein coding

ENSMUSP00000117... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Tumour protein D52 PANTHER Tumour protein D52

PTHR19307:SF13

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

Variant Legend

missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 200 220

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

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