https://www.alphaknockout.com

Mouse Plp2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Plp2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Plp2 (NCBI Reference Sequence: NM_019755 ; Ensembl: ENSMUSG00000031146 ) is located on Mouse X. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000033486). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Plp2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-147H5 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: Males hemizygous for a null allele show increased neuronal apoptosis to ER stress and hypoxia in vitro and in a neonatal hypoxia-ischemia model in vivo. Mutant mouse embryonic fibroblasts show delayed ER trafficking, increased basal ER stress and increased sensitivity to ER stress-induced apoptosis.

Exon 2 starts from about 21.27% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1384 bp, and the size of intron 4 for 3'-loxP site insertion: 420 bp. The size of effective cKO region: ~1292 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 3 4 5 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Plp2 cKO region Exon of mouse Prickle3 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(7752bp) | A(24.28% 1882) | C(22.81% 1768) | T(24.68% 1913) | G(28.24% 2189)

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% chrX - 7670009 7673008 3000 browser details YourSeq 195 1668 1866 3000 99.0% chr13 - 97099142 97099340 199 browser details YourSeq 193 1668 1866 3000 98.5% chr2 + 75808255 75808453 199 browser details YourSeq 119 969 1167 3000 84.9% chr19 + 5035370 5035573 204 browser details YourSeq 118 948 1173 3000 83.2% chr19 + 61078070 61078300 231 browser details YourSeq 114 948 1168 3000 77.7% chr12 + 70194915 70195140 226 browser details YourSeq 113 948 1169 3000 83.8% chr15 + 101403280 101403498 219 browser details YourSeq 107 948 1143 3000 86.0% chr16 + 14134236 14134433 198 browser details YourSeq 106 952 1142 3000 81.5% chr7 - 120898396 120898584 189 browser details YourSeq 106 948 1127 3000 83.6% chr1 - 93084480 93084660 181 browser details YourSeq 106 948 1169 3000 82.4% chr12 + 103318848 103319060 213 browser details YourSeq 105 943 1143 3000 81.0% chr11 - 20709064 20709265 202 browser details YourSeq 100 948 1143 3000 78.7% chr19 - 32331519 32331713 195 browser details YourSeq 99 942 1121 3000 77.8% chr13 - 107227383 107227566 184 browser details YourSeq 99 969 1170 3000 83.3% chr11 - 49972876 49973074 199 browser details YourSeq 98 947 1143 3000 77.7% chr4 - 49224098 49224294 197 browser details YourSeq 98 948 1143 3000 77.0% chr12 - 88890956 88891149 194 browser details YourSeq 97 942 1121 3000 88.3% chr7 - 30051965 30052150 186 browser details YourSeq 95 951 1120 3000 78.3% chr6 + 120277416 120277584 169 browser details YourSeq 95 947 1169 3000 87.0% chr10 + 61502591 61502812 222

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% chrX - 7665717 7668716 3000 browser details YourSeq 560 210 773 3000 99.7% chr13 - 97098236 97098799 564 browser details YourSeq 550 210 773 3000 98.8% chr2 + 75808794 75809357 564 browser details YourSeq 258 298 757 3000 87.4% chr10 + 71483601 71484530 930 browser details YourSeq 80 330 529 3000 90.1% chr14 + 122180362 122459048 278687 browser details YourSeq 66 297 528 3000 92.3% chr1 - 10442784 10550761 107978 browser details YourSeq 63 330 524 3000 95.8% chrX - 137045820 137046215 396 browser details YourSeq 62 299 392 3000 89.9% chr4 - 127947385 127947511 127 browser details YourSeq 59 292 383 3000 86.6% chr11 + 120038405 120038498 94 browser details YourSeq 58 293 379 3000 85.2% chr15 + 58160574 58160662 89 browser details YourSeq 57 326 392 3000 98.4% chr10 + 121977227 121977325 99 browser details YourSeq 56 369 1331 3000 95.1% chr1 - 136426577 136698138 271562 browser details YourSeq 53 331 527 3000 84.5% chr10 - 60021778 60022168 391 browser details YourSeq 52 293 369 3000 84.5% chr11 - 115304311 115304388 78 browser details YourSeq 51 294 369 3000 79.8% chr15 + 90175435 90175508 74 browser details YourSeq 50 293 370 3000 82.4% chr10 - 28418806 28418881 76 browser details YourSeq 49 327 382 3000 96.5% chr14 - 20680499 20680589 91 browser details YourSeq 49 293 369 3000 81.9% chr14 + 49192460 49192536 77 browser details YourSeq 48 330 383 3000 96.2% chr11 - 75602796 75602851 56 browser details YourSeq 47 294 362 3000 84.1% chr1 - 118479929 118479997 69

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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

Gene and information: Plp2 proteolipid protein 2 [ Mus musculus (house mouse) ] Gene ID: 18824, updated on 12-Aug-2019

Gene summary

Official Symbol Plp2 provided by MGI Official Full Name proteolipid protein 2 provided by MGI Primary source MGI:MGI:1298382 See related Ensembl:ENSMUSG00000031146 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 mIMA4 Expression Broad expression in genital fat pad adult (RPKM 122.4), colon adult (RPKM 120.2) and 23 other tissues See more Orthologs human all

Genomic context

Location: X A1.1; X 3.45 cM See Plp2 in Genome Data Viewer

Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (7667941..7671390, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (7245242..7248404, complement)

Chromosome X - NC_000086.7

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

Transcript information: This gene has 3 transcripts

Gene: Plp2 ENSMUSG00000031146

Description proteolipid protein 2 [Source:MGI Symbol;Acc:MGI:1298382] Gene Synonyms mIMA4 Location Chromosome X: 7,667,941-7,671,390 reverse strand. GRCm38:CM001013.2 About this gene This gene has 3 transcripts (splice variants), 185 orthologues, 17 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Plp2-201 ENSMUST00000033486.5 1157 152aa ENSMUSP00000033486.5 Protein coding CCDS40842 Q0VEW4 Q9R1Q7 TSL:1 GENCODE basic APPRIS P1

Plp2-202 ENSMUST00000136155.1 1212 No protein - Retained intron - - TSL:2

Plp2-203 ENSMUST00000143854.1 600 No protein - Retained intron - - TSL:2

23.45 kb Forward strand

7.66Mb 7.67Mb 7.68Mb Prickle3-201 >protein coding (Comprehensive set...

Prickle3-204 >retained intron Prickle3-202 >retained intron

Prickle3-203 >protein coding

Prickle3-205 >retained intron

Contigs AL672231.7 >

Genes (Comprehensive set... < Plp2-201protein coding < Magix-202lncRNA

< Plp2-202retained intron < Magix-201protein coding

< Plp2-203retained intron

Regulatory Build

7.66Mb 7.67Mb 7.68Mb Reverse strand 23.45 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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

Transcript: ENSMUST00000033486

< Plp2-201protein coding

Reverse strand 3.45 kb

ENSMUSP00000033... Transmembrane heli... Low complexity (Seg) Pfam Marvel domain PROSITE profiles Marvel domain PANTHER PTHR22776

PTHR22776:SF4

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 152

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

Page 7 of 7