Mouse Abcg2 Conditional Knockout Project (CRISPR/Cas9) · 2020. 12. 2. · Gene ID: 26357, updated on 24-Oct-2019 Gene summary Genomic context Exon count: 22 Annotation release Status

Mouse Abcg2 Conditional Knockout Project(CRISPR/Cas9)

Objective:To create a Abcg2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary:The Abcg2 gene (NCBI Reference Sequence: NM_011920.3 ; Ensembl: ENSMUSG00000029802 ) is located onMouse chromosome 6.16 exons are identified , with the ATG start codon in exon 2 and the TAA stop codon in exon 16 (Transcript:ENSMUST00000031822).Exon 3~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in theloss of function of the Mouse Abcg2 gene.To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC cloneRP23-46D10 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: Homozygous null mice exhibit extreme sensitivity to the dietary chlorophyll-catabolite pheophorbide a, resulting insevere phototoxic skin lesions upon light exposure. Mutants show a novel form of protoporphyria, associated with a 10-fold increase in erythrocyte levels of protoporphyrin IX.

Exon 3 starts from about 10.2% of the coding region. The knockout of Exon 3~5 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 7757 bp, and the size of intron 5 for 3'-loxP site insertion: 1625 bp. The size of effective cKO region: ~2395 bp.This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level.

https://www.alphaknockout.com

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https://www.ncbi.nlm.nih.gov/clone?term=RP23-46D10https://www.alphaknockout.com

Overview of the Targeting Strategy

gRNA region gRNA region

1 3 4 5 6 16

Wildtype allele5' 3'

Targeting vector

Targeted allele

Constitutive KO allele(After Cre recombination)

Legends Exon of mouse Abcg2 Homology arm cKO region loxP site


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Overview of the Dot Plot

Overview of the GC Content Distribution

Sequence 1Sequence 2

Reverse ComplementForward

Window size: 10 bp

Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. Tandem repeats are foundin the dot plot matrix. It may be difficult to construct this targeting vector.

Sequence 1Sequence 2

Window size: 300 bp

Summary: Full Length(8643bp) | A(26.81% 2317) | C(18.59% 1607) | T(31.81% 2749) | G(22.79% 1970)

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% chr6 + 58660365 58663364 3000browser details YourSeq 196 1 293 3000 83.9% chrX + 106690392 106690684 293browser details YourSeq 194 1 293 3000 84.4% chr9 - 85881936 85882228 293browser details YourSeq 187 1 293 3000 82.7% chr13 - 35105055 35105348 294browser details YourSeq 186 1 293 3000 86.2% chr1 + 66582789 66583084 296browser details YourSeq 182 1 276 3000 83.8% chr8 + 46134921 46135197 277browser details YourSeq 179 34 289 3000 85.2% chr5 - 25568176 25568432 257browser details YourSeq 177 34 291 3000 84.5% chr7 - 18306902 18307159 258browser details YourSeq 174 1 261 3000 81.6% chr19 - 34801092 34801340 249browser details YourSeq 174 1 293 3000 84.6% chr10 - 82211178 82211471 294browser details YourSeq 174 1 293 3000 86.2% chr1 + 16383523 16383816 294browser details YourSeq 173 1 295 3000 85.5% chr18 - 32502688 32502983 296browser details YourSeq 173 1 258 3000 83.8% chr10 + 130520783 130521041 259browser details YourSeq 168 1 247 3000 85.0% chr8 - 75357697 75357944 248browser details YourSeq 165 11 267 3000 82.9% chr7 - 27068975 27069232 258browser details YourSeq 165 1 267 3000 83.3% chr11 - 58448190 58448655 466browser details YourSeq 163 1 258 3000 82.0% chr17 - 53352633 53352891 259browser details YourSeq 162 1 293 3000 80.2% chr5 - 83116396 83116683 288browser details YourSeq 160 34 293 3000 83.7% chrX - 135116630 135116890 261browser details YourSeq 160 11 293 3000 84.0% chr5 + 24151854 24152136 283

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% chr6 + 58665508 58668507 3000browser details YourSeq 148 594 773 3000 90.8% chr11 + 59128642 59128807 166browser details YourSeq 144 594 773 3000 96.3% chr2 + 91316389 91316575 187browser details YourSeq 144 455 777 3000 91.1% chr14 + 19362779 19363122 344browser details YourSeq 143 559 768 3000 88.3% chr4 - 74306655 74306846 192browser details YourSeq 139 594 867 3000 86.3% chr3 + 94743835 94744068 234browser details YourSeq 138 594 773 3000 90.5% chrX + 60492387 60492559 173browser details YourSeq 137 594 773 3000 86.5% chr7 + 80834434 80834596 163browser details YourSeq 137 594 773 3000 90.4% chr12 + 84666260 84666431 172browser details YourSeq 137 594 773 3000 86.5% chr11 + 52348523 52348685 163browser details YourSeq 136 594 777 3000 90.7% chr2 - 38928191 38928372 182browser details YourSeq 136 594 762 3000 92.0% chr17 - 13546143 13546334 192browser details YourSeq 135 597 766 3000 88.6% chr2 - 130036300 130036460 161browser details YourSeq 133 594 737 3000 96.6% chr10 - 71134028 71134174 147browser details YourSeq 133 594 742 3000 94.7% chr2 + 28557118 28557266 149browser details YourSeq 133 594 761 3000 91.9% chr11 + 35132387 35132570 184browser details YourSeq 132 594 768 3000 92.4% chr12 - 80638040 80638246 207browser details YourSeq 132 594 766 3000 87.9% chr17 + 24553712 24553876 165browser details YourSeq 131 594 773 3000 84.6% chr6 - 120683228 120683390 163browser details YourSeq 131 594 742 3000 94.0% chr6 + 51596878 51597026 149

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.


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Official Symbol Abcg2 provided by MGIOfficial Full Name ATP binding cassette subfamily G member 2 (Junior blood group) provided by MGI

Primary source MGI:MGI:1347061See related Ensembl:ENSMUSG00000029802Gene type protein coding

RefSeq status VALIDATEDOrganism Mus musculus

Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires;Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus

Also known as MXR; ABCP; BCRP; MXR1; ABC15; Bcrp1; AI428558Summary The membrane-associated protein encoded by this gene is included in the superfamily of ATP-binding cassette (ABC)

transporters. ABC proteins transport various molecules across extra- and intra-cellular membranes. ABC genes are dividedinto seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the Whitesubfamily. Alternatively referred to as a breast cancer resistance protein, the human protein functions as a xenobiotictransporter which may play a major role in multi-drug resistance. This protein likely serves as a cellular defense mechanismin response to mitoxantrone and anthracycline exposure. [provided by RefSeq, Jul 2008]

Expression

Broad expression in kidney adult (RPKM 44.7), placenta adult (RPKM 44.0) and 23 other tissues See moreOrthologshuman all

See Abcg2 in Genome Data ViewerLocation: 6 B3; 6 27.82 cM

Gene and protein information:Abcg2 ATP binding cassette subfamily G member 2 (Junior blood group) [ Mus musculus (house mouse) ]Gene ID: 26357, updated on 24-Oct-2019

Gene summary

Genomic context

Exon count: 22

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (58584491..58692451)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (58546666..58642445)

Chromosome 6 - NC_000072.6


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http://www.informatics.jax.org/mgihome/nomen/http://www.informatics.jax.org/mgihome/nomen/http://www.informatics.jax.org/marker/MGI:1347061http://www.ensembl.org/id/ENSMUSG00000029802https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10090https://www.ncbi.nlm.nih.gov/gene/9429https://www.ncbi.nlm.nih.gov/gene/9429/ortholog/https://www.ncbi.nlm.nih.gov/genome/gdv/?context=gene&acc=26357https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Mus_musculus/108/https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.26/https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.18/https://www.alphaknockout.com

Description ATP binding cassette subfamily G member 2 (Junior blood group) [Source:MGI Symbol;Acc:MGI:1347061]Gene Synonyms Bcrp

Location Chromosome 6: 58,584,523-58,695,676 forward strand.GRCm38:CM000999.2

About this gene This gene has 13 transcripts (splice variants), 224 orthologues, 5 paralogues, is a member of 1 Ensembl protein family andis associated with 28 phenotypes.

Transcripts

Transcript information: This gene has 13 transcripts

Gene: Abcg2 ENSMUSG00000029802

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Abcg2-201

ENSMUST00000031822.12 5717 657aa ENSMUSP00000031822.6 Protein coding CCDS20195 A0A0R4J0B6 TSL:1GENCODEbasicAPPRIS P1

Abcg2-202

ENSMUST00000114294.7 2178 657aa ENSMUSP00000109933.1 Protein coding CCDS20195 A0A0R4J0B6 TSL:5GENCODEbasicAPPRIS P1

Abcg2-213

ENSMUST00000204948.1 664 172aa ENSMUSP00000144876.1 Protein coding - D3Z150 CDS 3'incompleteTSL:2

Abcg2-207

ENSMUST00000145161.7 603 172aa ENSMUSP00000122924.1 Protein coding - D3Z150 CDS 3'incompleteTSL:2

Abcg2-211

ENSMUST00000203146.2 562 159aa ENSMUSP00000145435.1 Protein coding - A0A0N4SWA2 CDS 3'incompleteTSL:5

Abcg2-209

ENSMUST00000147213.7 464 115aa ENSMUSP00000120940.1 Protein coding - D3YZX5 CDS 3'incompleteTSL:5

Abcg2-210

ENSMUST00000148008.1 348 50aa ENSMUSP00000114454.1 Protein coding - D3YVG8 CDS 3'incompleteTSL:3

Abcg2-206

ENSMUST00000143752.7 2556 576aa ENSMUSP00000138608.1 Nonsense mediateddecay

- S4R2E1 TSL:5

Abcg2-208

ENSMUST00000145685.1 600 130aa ENSMUSP00000138703.1 Nonsense mediateddecay

- S4R2M4 CDS 5'incompleteTSL:5

Abcg2-204

ENSMUST00000134426.1 1133 Noprotein

- Retained intron - - TSL:1

Abcg2-203



Abcg2-205



Abcg2-212


- lncRNA - - TSL:1


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http://www.informatics.jax.org/marker/MGI:1347061http://www.ensembl.org/Mus_musculus/Location/View?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676http://www.ensembl.org/Mus_musculus/Gene/Splice?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676http://www.ensembl.org/Mus_musculus/Gene/Compara_Ortholog?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676http://www.ensembl.org/Mus_musculus/Gene/Compara_Paralog?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676http://www.ensembl.org/Mus_musculus/Gene/Family?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676http://www.ensembl.org/Mus_musculus/Gene/Phenotype?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000031822http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000031822http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000031822http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS20195http://www.uniprot.org/uniprot/A0A0R4J0B6http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000114294http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000114294http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000114294http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS20195http://www.uniprot.org/uniprot/A0A0R4J0B6http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000204948http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000204948http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000204948http://www.uniprot.org/uniprot/D3Z150http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000145161http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000145161http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000145161http://www.uniprot.org/uniprot/D3Z150http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000203146http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000203146http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000203146http://www.uniprot.org/uniprot/A0A0N4SWA2http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000147213http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000147213http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000147213http://www.uniprot.org/uniprot/D3YZX5http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000148008http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000148008http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000148008http://www.uniprot.org/uniprot/D3YVG8http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000143752http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000143752http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000143752http://www.uniprot.org/uniprot/S4R2E1http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000145685http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000145685http://www.ensembl.org/Mus_musculus/Transcript/ProteinSummary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000145685http://www.uniprot.org/uniprot/S4R2M4http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000134426http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000124542http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000143447http://www.ensembl.org/Mus_musculus/Transcript/Summary?db=core;g=ENSMUSG00000029802;r=6:58584523-58695676;t=ENSMUST00000204924https://www.alphaknockout.com

58.60Mb 58.65Mb 58.70Mb

Abcg2-209 >protein coding Abcg2-208 >nonsense mediated decay

Abcg2-212 >lncRNA Abcg2-203 >retained intron Abcg2-204 >retained intron

Abcg2-210 >protein coding Abcg2-205 >retained intron

Abcg2-201 >protein coding

Abcg2-206 >nonsense mediated decay





Genes(Comprehensive set...

AC121855.3 > < AC121859.2Contigs

Regulatory Build

58.60Mb 58.65Mb 58.70Mb

CTCF

Enhancer

Open Chromatin

Promoter

Promoter Flank

Transcription Factor Binding Site

Regulation Legend

Protein Coding

merged Ensembl/Havana

Ensembl protein coding

Non-Protein Coding

processed transcript

RNA gene

Gene Legend

131.15 kb Forward strand

Reverse strand 131.15 kb


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Transcript: ENSMUST00000031822


99.01 kb Forward strand

ENSMUSP00000031...Transmembrane heli...Low complexity (Seg)

P-loop containing nucleoside triphosphate hydrolaseSuperfamily

AAA+ ATPase domainSMART

ABC transporter-like ABC-2 type transporterPfam

ABC transporter-likePROSITE profiles

ATP-binding cassette subfamily G member 2

PTHR19241

PANTHER

3.40.50.300Gene3D

cd03213CDD

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

missense variant

synonymous variant

Variant Legend

0 60 120 180 240 300 360 420 480 5400 657Scale bar

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


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Mouse Abcg2 Conditional Knockout Project (CRISPR/Cas9)The preview version strategy is for reference only, please contact our salesperson for the final version strategy if you do order from us.Objective:To create a Abcg2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary:Note: Homozygous null mice exhibit extreme sensitivity to the dietary chlorophyll-catabolite pheophorbide a, resulting in severe phototoxic skin lesions upon light exposure. Mutants show a novel form of protoporphyria, associated with a 10-fold increase in erythrocyte levels of protoporphyrin IX.

Overview of the Targeting StrategyOverview of the Dot PlotOverview of the GC Content DistributionBLAT Search Results (up)BLAT Search Results (down)Gene and protein information:

Abcg2 ATP binding cassette subfamily G member 2 (Junior blood group) [ Mus musculus (house mouse) ]Gene summaryGenomic contextTranscript information: This gene has 13 transcriptsTranscript information: This gene has 13 transcripts

Gene: Abcg2 ENSMUSG00000029802Transcript: ENSMUST00000031822Transcript: ENSMUST00000031822

Documents

Mouse Abcg2 Conditional Knockout Project (CRISPR/Cas9) · 2020. 12. 2. · Gene ID: 26357, updated on 24-Oct-2019 Gene summary Genomic context Exon count: 22 Annotation release Status