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Mouse Plcd3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Plcd3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Plcd3 gene (NCBI Reference Sequence: NM_152813 ; Ensembl: ENSMUSG00000020937 ) is located on Mouse chromosome 11. 15 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 15 (Transcript: ENSMUST00000103077). Exon 9~10 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Plcd3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-133L3 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 9 starts from about 59.53% of the coding region. The knockout of Exon 9~10 will result in frameshift of the gene. The size of intron 8 for 5'-loxP site insertion: 1710 bp, and the size of intron 10 for 3'-loxP site insertion: 714 bp. The size of effective cKO region: ~945 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 8 9 10 11 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Plcd3 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(7445bp) | A(22.28% 1659) | C(26.68% 1986) | T(23.99% 1786) | G(27.05% 2014)

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% chr11 - 103075254 103078253 3000 browser details YourSeq 226 1711 2247 3000 83.5% chr2 + 91740324 91740679 356 browser details YourSeq 223 1737 2246 3000 90.0% chr2 - 168345659 168759129 413471 browser details YourSeq 210 1723 2245 3000 86.1% chr1 + 180731675 180731994 320 browser details YourSeq 202 1724 2245 3000 92.1% chr15 - 62021500 62100481 78982 browser details YourSeq 191 1725 2248 3000 84.7% chr19 + 37261667 37262172 506 browser details YourSeq 186 1741 2247 3000 83.4% chr16 - 13601716 13602076 361 browser details YourSeq 165 1710 2200 3000 88.5% chr19 - 6327806 6328291 486 browser details YourSeq 158 1737 2244 3000 87.0% chr4 + 150573348 150573690 343 browser details YourSeq 155 1710 2168 3000 92.4% chr4_JH584294_random + 123490 123986 497 browser details YourSeq 155 1710 2168 3000 92.4% chr4 + 42775695 42776191 497 browser details YourSeq 153 1710 1876 3000 96.5% chr7 - 16992964 16993134 171 browser details YourSeq 151 1714 1874 3000 97.6% chr11 - 100839190 100839390 201 browser details YourSeq 150 1711 1874 3000 96.4% chr12 - 112901126 112901293 168 browser details YourSeq 148 1701 1877 3000 92.6% chr14 - 101723493 101723692 200 browser details YourSeq 147 1710 1874 3000 93.9% chr3 - 9043126 9043289 164 browser details YourSeq 146 1715 1875 3000 94.3% chr4 - 124878490 124878647 158 browser details YourSeq 146 1710 1874 3000 94.6% chr2 - 136857315 136857480 166 browser details YourSeq 146 1710 1875 3000 95.1% chr2 + 137051929 137052098 170 browser details YourSeq 145 1706 1871 3000 95.7% chr17 + 29177381 29177548 168

Note: The 3000 bp section upstream of Exon 9 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% chr11 - 103071309 103074308 3000 browser details YourSeq 202 656 1162 3000 85.7% chr7 - 4908555 4908939 385 browser details YourSeq 190 652 1161 3000 83.4% chr6 - 31039839 31040173 335 browser details YourSeq 176 683 1162 3000 82.7% chr7 + 128758546 128758857 312 browser details YourSeq 172 654 850 3000 93.7% chr11 + 91299585 91299778 194 browser details YourSeq 169 652 1085 3000 83.6% chr13 - 74516466 74516752 287 browser details YourSeq 165 616 841 3000 87.6% chr5 - 23646718 23646925 208 browser details YourSeq 165 664 887 3000 93.7% chr9 + 92259533 92259778 246 browser details YourSeq 163 664 1162 3000 84.1% chr16 - 32308460 32308790 331 browser details YourSeq 162 659 846 3000 93.1% chr18 + 6460527 6460714 188 browser details YourSeq 158 656 850 3000 89.8% chr5 + 129944427 129944617 191 browser details YourSeq 156 658 841 3000 91.5% chr6 - 126796663 126796839 177 browser details YourSeq 156 664 1162 3000 80.6% chr7 + 3363066 3363342 277 browser details YourSeq 155 657 850 3000 89.1% chr17 - 27693535 27693721 187 browser details YourSeq 155 656 850 3000 91.9% chr14 - 54422598 54422794 197 browser details YourSeq 155 656 850 3000 90.2% chr9 + 62150487 62150678 192 browser details YourSeq 155 656 850 3000 91.8% chr12 + 111799790 111799983 194 browser details YourSeq 155 651 850 3000 89.4% chr10 + 120654797 120654993 197 browser details YourSeq 154 662 850 3000 93.3% chr13 - 42141636 42142003 368 browser details YourSeq 154 652 850 3000 91.4% chr5 + 23747035 23747238 204

Note: The 3000 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Plcd3 phospholipase C, delta 3 [ Mus musculus (house mouse) ] Gene ID: 72469, updated on 10-Oct-2019

Gene summary

Official Symbol Plcd3 provided by MGI Official Full Name phospholipase C, delta 3 provided by MGI Primary source MGI:MGI:107451 See related Ensembl:ENSMUSG00000020937 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 mKIAA1964; 2610205J15Rik Expression Broad expression in bladder adult (RPKM 8.4), stomach adult (RPKM 7.5) and 25 other tissues See more Orthologs human all

Genomic context

Location: 11 E1; 11 66.71 cM See Plcd3 in Genome Data Viewer

Exon count: 18

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (103070296..103101710, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (102931610..102962972, complement)

Chromosome 11 - NC_000077.6

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Transcript information: This gene has 5 transcripts

Gene: Plcd3 ENSMUSG00000020937

Description phospholipase C, delta 3 [Source:MGI Symbol;Acc:MGI:107451] Gene Synonyms 2610205J15Rik Location Chromosome 11: 103,070,304-103,101,658 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 263 orthologues, 14 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Plcd3-201 ENSMUST00000103077.1 3023 785aa ENSMUSP00000099366.1 Protein coding CCDS25512 Q8K2J0 TSL:1 GENCODE basic APPRIS P1

Plcd3-203 ENSMUST00000128650.7 1952 471aa ENSMUSP00000117455.1 Protein coding - F7AAE0 CDS 5' incomplete TSL:1

Plcd3-202 ENSMUST00000124740.1 591 No protein - lncRNA - - TSL:5

Plcd3-205 ENSMUST00000143012.1 509 No protein - lncRNA - - TSL:5

Plcd3-204 ENSMUST00000142970.1 425 No protein - lncRNA - - TSL:2

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51.35 kb Forward strand 103.07Mb 103.08Mb 103.09Mb 103.10Mb 103.11Mb Genes Nmt1-201 >protein coding Acbd4-202 >nonsense mediated decay (Comprehensive set...

Acbd4-205 >nonsense mediated decay

Acbd4-203 >protein coding

Acbd4-209 >protein coding

Acbd4-206 >lncRNA

Acbd4-201 >protein coding

Acbd4-211 >retained intron

Acbd4-207 >protein coding

Acbd4-216 >retained intron

Acbd4-208 >lncRNA

Acbd4-214 >protein coding

Acbd4-212 >retained intron

Acbd4-210 >retained intron

Acbd4-213 >retained intron

Acbd4-215 >lncRNA

Acbd4-204 >lncRNA

Contigs AL731805.8 >

Genes (Comprehensive set... < Plcd3-201protein coding

< Plcd3-203protein coding < Plcd3-205lncRNA

< Plcd3-202lncRNA < Plcd3-204lncRNA

Regulatory Build

103.07Mb 103.08Mb 103.09Mb 103.10Mb 103.11Mb Reverse strand 51.35 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000103077

< Plcd3-201protein coding

Reverse strand 31.36 kb

ENSMUSP00000099... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 EF-hand domain pair PLC-like phosphodiesterase, TIM beta/alpha-barrel domain superfamily

SSF49562 SMART Pleckstrin homology domain Phosphatidylinositol-specific phospholipase C, X domain

Phospholipase C, phosphatidylinositol-specific, Y domain

C2 domain Prints Phosphoinositide phospholipase C family Pfam C2 domain

1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-3, EF hand domain

Phosphatidylinositol-specific phospholipase C, X domain

Phospholipase C, phosphatidylinositol-specific, Y domain PROSITE profiles PS50007 C2 domain

Phospholipase C, phosphatidylinositol-specific, Y domain PANTHER 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-3

Phosphoinositide phospholipase C family Gene3D 1.10.238.10 PLC-like phosphodiesterase, TIM beta/alpha-barrel domain superfamily

PH-like domain superfamily C2 domain superfamily CDD cd13363 cd08630 cd00275

1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-3, EF hand domain

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 785

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

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