https://www.alphaknockout.com

Mouse Vpreb3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Vpreb3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Vpreb3 gene (NCBI Reference Sequence: NM_009514 ; Ensembl: ENSMUSG00000000903 ) is located on Mouse chromosome 10. 2 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000000926). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Vpreb3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-69G9 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 2 covers 85.91% of the coding region. Start codon is in exon 1, and stop codon is in exon 2. The size of intron 1 for 5'-loxP site insertion: 733 bp. The size of effective cKO region: ~1265 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Vpreb3 cKO region loxP site

Page 2 of 7 https://www.alphaknockout.com

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(6817bp) | A(25.06% 1708) | C(24.39% 1663) | T(25.14% 1714) | G(25.41% 1732)

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.

Page 3 of 7 https://www.alphaknockout.com

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 + 75945894 75948893 3000 browser details YourSeq 416 1605 2192 3000 87.3% chr13 + 60017428 60018004 577 browser details YourSeq 409 1605 2192 3000 88.0% chr7 + 121662650 121663232 583 browser details YourSeq 404 1602 2192 3000 86.7% chr5 - 128310813 128311397 585 browser details YourSeq 403 1241 2172 3000 85.0% chr6 + 11820973 11821776 804 browser details YourSeq 399 1602 2192 3000 85.1% chr4 + 31695075 31695636 562 browser details YourSeq 398 1597 2171 3000 86.4% chr13 - 36600205 36600773 569 browser details YourSeq 397 1605 2192 3000 85.2% chr13 - 94041922 94042501 580 browser details YourSeq 396 1605 2192 3000 84.8% chr6 + 134272187 134272785 599 browser details YourSeq 394 1605 2172 3000 87.1% chr17 + 12096340 12096901 562 browser details YourSeq 393 1605 2142 3000 88.0% chr10 + 70489622 70490153 532 browser details YourSeq 392 1624 2192 3000 85.9% chr11 - 103025154 103025713 560 browser details YourSeq 390 1605 2192 3000 85.6% chr13 - 69684107 69684701 595 browser details YourSeq 385 1625 2191 3000 86.4% chr5 + 144050421 144050976 556 browser details YourSeq 381 1605 2192 3000 84.1% chr18 - 73848418 73849013 596 browser details YourSeq 381 1600 2166 3000 87.8% chr1 - 89160482 89161043 562 browser details YourSeq 379 1627 2192 3000 85.1% chr5 + 100122577 100123124 548 browser details YourSeq 379 1605 2172 3000 85.7% chr10 + 122100375 122100920 546 browser details YourSeq 375 1605 2171 3000 85.4% chr4 - 136024849 136025411 563 browser details YourSeq 375 1605 2193 3000 88.0% chr2 + 163945666 163946251 586

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% chr10 + 75949461 75952460 3000 browser details YourSeq 146 783 2830 3000 91.5% chr11 - 96232285 96441406 209122 browser details YourSeq 131 798 2831 3000 93.4% chr13 + 29952515 30358102 405588 browser details YourSeq 124 2697 2890 3000 94.4% chr1 + 131941999 131942563 565 browser details YourSeq 120 2696 2831 3000 94.9% chr1 + 153547093 153547251 159 browser details YourSeq 116 2696 2831 3000 94.7% chr14 - 52179736 52179916 181 browser details YourSeq 114 2703 2831 3000 96.0% chr14 - 55867515 55867674 160 browser details YourSeq 112 2703 2830 3000 96.0% chr11 - 76603370 76603519 150 browser details YourSeq 112 2696 2831 3000 93.2% chr4 + 155431868 155432212 345 browser details YourSeq 112 2703 2831 3000 96.0% chr15 + 65228081 65228231 151 browser details YourSeq 112 2703 2831 3000 95.2% chr15 + 36763028 36763178 151 browser details YourSeq 111 809 2830 3000 97.5% chr17 - 46695450 46807121 111672 browser details YourSeq 111 2696 2831 3000 93.1% chr15 - 71451555 71451729 175 browser details YourSeq 111 2703 2830 3000 94.4% chr13 - 104836609 104836760 152 browser details YourSeq 110 2697 2831 3000 93.7% chr10 - 82656429 82656583 155 browser details YourSeq 110 2703 2857 3000 89.9% chr11 + 86372395 86372572 178 browser details YourSeq 109 2704 2830 3000 95.1% chr14 + 65862364 65862512 149 browser details YourSeq 108 2705 2831 3000 94.3% chr18 - 37788430 37788577 148 browser details YourSeq 108 2703 2832 3000 95.0% chr17 - 15889389 15889534 146 browser details YourSeq 108 2696 2830 3000 92.2% chr11 - 94611771 94611928 158

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 https://www.alphaknockout.com

Gene and protein information: Vpreb3 pre-B lymphocyte gene 3 [ Mus musculus (house mouse) ] Gene ID: 22364, updated on 12-Aug-2019

Gene summary

Official Symbol Vpreb3 provided by MGI Official Full Name pre-B lymphocyte gene 3 provided by MGI Primary source MGI:MGI:98938 See related Ensembl:ENSMUSG00000000903 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 8HS-20; Vpreb-3; AI528709 Expression Biased expression in spleen adult (RPKM 93.8), ovary adult (RPKM 27.8) and 6 other tissues See more Orthologs human all

Genomic context

Location: 10; 10 C1 See Vpreb3 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (75943024..75949657)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (75411057..75412391)

Chromosome 10 - NC_000076.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 2 transcripts

Gene: Vpreb3 ENSMUSG00000000903

Description pre-B lymphocyte gene 3 [Source:MGI Symbol;Acc:MGI:98938] Gene Synonyms 8HS-20, Vpreb-3 Location Chromosome 10: 75,943,057-75,949,657 forward strand. GRCm38:CM001003.2 About this gene This gene has 2 transcripts (splice variants), 117 orthologues, 145 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Vpreb3-201 ENSMUST00000000926.2 625 123aa ENSMUSP00000000926.2 Protein coding CCDS23940 Q61243 TSL:1 GENCODE basic APPRIS P1

Vpreb3-202 ENSMUST00000121151.1 729 130aa ENSMUSP00000113205.1 Protein coding - D3Z6J4 TSL:3 GENCODE basic

26.60 kb Forward strand 75.935Mb 75.940Mb 75.945Mb 75.950Mb 75.955Mb Genes Chchd10-203 >protein coding Vpreb3-202 >protein coding Gm5134-201 >protein coding (Comprehensive set...

Chchd10-201 >protein coding Vpreb3-201 >protein coding

Chchd10-202 >retained intron

Contigs < AC134382.13 Genes < Mmp11-206protein coding < Gm867-201lncRNA < Gm16221-201processed pseudogene (Comprehensive set...

< Gm867-202protein coding

Regulatory Build

75.935Mb 75.940Mb 75.945Mb 75.950Mb 75.955Mb Reverse strand 26.60 kb

Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript pseudogene

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000000926

1.36 kb Forward strand

Vpreb3-201 >protein coding

ENSMUSP00000000... Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily

SMART Immunoglobulin V-set domain Pfam Immunoglobulin V-set domain PROSITE profiles Immunoglobulin-like domain

PANTHER PTHR23267

PTHR23267:SF133 Gene3D Immunoglobulin-like fold

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 123

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

Page 7 of 7