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Mouse Shc2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Shc2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Shc2 gene (NCBI Reference Sequence: NM_001024539 ; Ensembl: ENSMUSG00000020312 ) is located on Mouse chromosome 10. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000020564). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Shc2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-382A7 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: Mice homozygous for disruptions in this gene display sensory nerve defects related to nociception.

Exon 3 starts from about 27.57% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1136 bp, and the size of intron 4 for 3'-loxP site insertion: 2574 bp. The size of effective cKO region: ~806 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 2 3 4 13 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Shc2 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(7306bp) | A(21.54% 1574) | C(27.35% 1998) | T(25.88% 1891) | G(25.23% 1843)

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% chr10 - 79630379 79633378 3000 browser details YourSeq 49 2584 2781 3000 68.8% chr12 - 23807591 23807708 118 browser details YourSeq 39 2160 2244 3000 95.4% chr12 - 3354114 3354263 150 browser details YourSeq 37 1484 1621 3000 89.8% chr3 - 108634187 108634322 136 browser details YourSeq 37 2731 2781 3000 86.3% chr1 - 37268995 37269045 51 browser details YourSeq 37 1484 1621 3000 97.5% chr16 + 16788802 16788941 140 browser details YourSeq 36 2160 2211 3000 92.9% chr14 - 14702221 14702273 53 browser details YourSeq 36 1484 1623 3000 95.2% chr12 - 99766839 99766982 144 browser details YourSeq 36 2730 2781 3000 90.7% chr11 + 62976604 62976657 54 browser details YourSeq 35 2132 2186 3000 87.2% chr12 - 72524120 72524172 53 browser details YourSeq 35 2707 2781 3000 87.2% chr17 + 5731191 5731263 73 browser details YourSeq 35 2722 2766 3000 88.9% chr1 + 183637498 183637542 45 browser details YourSeq 34 2730 2781 3000 84.1% chr12 - 22626631 22626681 51 browser details YourSeq 34 2730 2781 3000 84.1% chr12 - 21787636 21787686 51 browser details YourSeq 34 819 888 3000 94.9% chr15 + 98973995 98974372 378 browser details YourSeq 34 2730 2781 3000 84.1% chr12 + 19384566 19384616 51 browser details YourSeq 33 1492 1531 3000 92.5% chr16 - 94537776 94537816 41 browser details YourSeq 32 811 849 3000 92.4% chr16 + 38531164 38531203 40 browser details YourSeq 32 1193 1224 3000 100.0% chr15 + 66441879 66441910 32 browser details YourSeq 31 2751 2781 3000 100.0% chr19 - 55684055 55684085 31

Note: The 3000 bp section upstream of Exon 3 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% chr10 - 79626573 79629572 3000 browser details YourSeq 214 1272 1533 3000 92.3% chrX - 153677973 153678267 295 browser details YourSeq 210 1272 1536 3000 93.9% chr5 + 120832681 120833006 326 browser details YourSeq 209 1270 1533 3000 91.1% chr8 + 83950769 83951082 314 browser details YourSeq 201 1260 1533 3000 87.0% chr16 + 32223494 32223757 264 browser details YourSeq 200 1272 1533 3000 90.1% chr6 + 129360699 129360972 274 browser details YourSeq 199 1267 1533 3000 88.6% chr9 + 88553664 88553947 284 browser details YourSeq 198 1095 1516 3000 84.6% chr11 - 117795501 117795773 273 browser details YourSeq 198 1272 1533 3000 91.7% chr15 + 98716675 98716948 274 browser details YourSeq 197 1272 1533 3000 88.5% chr2 - 32114751 32115007 257 browser details YourSeq 197 1272 1533 3000 91.7% chr14 - 120954373 120954661 289 browser details YourSeq 197 1272 1533 3000 89.1% chr5 + 65798805 65799099 295 browser details YourSeq 197 1276 1533 3000 92.4% chr17 + 29364220 29364509 290 browser details YourSeq 196 1273 1529 3000 93.5% chr2 - 119409886 119410143 258 browser details YourSeq 196 1272 1533 3000 93.1% chr12 + 21509391 21509668 278 browser details YourSeq 195 1272 1533 3000 91.6% chr17 - 84069439 84069711 273 browser details YourSeq 195 1272 1533 3000 93.4% chr8 + 117289796 117290087 292 browser details YourSeq 194 1270 1533 3000 92.7% chr3 - 116747742 116748012 271 browser details YourSeq 194 1272 1533 3000 91.2% chr3 - 88589608 88828245 238638 browser details YourSeq 193 1276 1533 3000 90.4% chr2 + 120694142 121126441 432300

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 protein information: Shc2 SHC (Src homology 2 domain containing) transforming protein 2 [ Mus musculus (house mouse) ] Gene ID: 216148, updated on 10-Oct-2019

Gene summary

Official Symbol Shc2 provided by MGI Official Full Name SHC (Src homology 2 domain containing) transforming protein 2 provided by MGI Primary source MGI:MGI:106180 See related Ensembl:ENSMUSG00000020312 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 SCK; Sli; ShcB; 6720466E06 Expression Broad expression in CNS E18 (RPKM 14.0), whole brain E14.5 (RPKM 11.3) and 19 other tissues See more Orthologs human all

Genomic context

Location: 10 C1; 10 39.72 cM See Shc2 in Genome Data Viewer

Exon count: 13

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (79617934..79637918, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (79080679..79100663, complement)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 3 transcripts

Gene: Shc2 ENSMUSG00000020312

Description SHC (Src homology 2 domain containing) transforming protein 2 [Source:MGI Symbol;Acc:MGI:106180] Gene Synonyms ShcB, Sli Location Chromosome 10: 79,618,051-79,637,918 reverse strand. GRCm38:CM001003.2 About this gene This gene has 3 transcripts (splice variants), 186 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Shc2-201 ENSMUST00000020564.6 3243 573aa ENSMUSP00000020564.6 Protein coding CCDS48620 E9QLZ0 Q8BMC3 TSL:5 GENCODE basic APPRIS P1

Shc2-203 ENSMUST00000168116.1 3720 No protein - Retained intron - - TSL:1

Shc2-202 ENSMUST00000166450.1 969 No protein - Retained intron - - TSL:3

39.87 kb Forward strand 79.61Mb 79.62Mb 79.63Mb 79.64Mb Genes Gm47163-201 >lncRNA (Comprehensive set...

Contigs AC132265.4 > Genes (Comprehensive set... < C2cd4c-201protein coding < Shc2-203retained intron < Odf3l2-201protein coding

< C2cd4c-202protein coding < Shc2-201protein coding

< Shc2-202retained intron

Regulatory Build

79.61Mb 79.62Mb 79.63Mb 79.64Mb Reverse strand 39.87 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: ENSMUST00000020564

< Shc2-201protein coding

Reverse strand 19.27 kb

ENSMUSP00000020... Low complexity (Seg) Superfamily SSF50729 SH2 domain superfamily

SMART PTB/PI domain SH2 domain

Prints Phosphotyrosine interaction domain, Shc-like SH2 domain Pfam PTB/PI domain SH2 domain

PROSITE profiles PTB/PI domain SH2 domain

PANTHER SHC-transforming protein 2

PTHR10337 Gene3D PH-like domain superfamily SH2 domain superfamily

CDD Phosphotyrosine interaction domain, Shc-like SH2 adaptor protein C, SH2 domain

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 573

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

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