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Mouse Mdh2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mdh2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mdh2 gene (NCBI Reference Sequence: NM_008617 ; Ensembl: ENSMUSG00000019179 ) is located on Mouse chromosome 5. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000019323). 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 Mdh2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-153M11 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 starts from about 6.61% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 4503 bp, and the size of intron 2 for 3'-loxP site insertion: 710 bp. The size of effective cKO region: ~864 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 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mdh2 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(7169bp) | A(25.28% 1812) | C(22.3% 1599) | T(25.9% 1857) | G(26.52% 1901)

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% chr5 + 135780056 135783055 3000 browser details YourSeq 76 1139 1271 3000 90.5% chr5 + 140368241 140698666 330426 browser details YourSeq 75 1176 1269 3000 91.4% chr5 + 140741602 140998891 257290 browser details YourSeq 68 1188 1271 3000 91.5% chr5 - 139127179 139127262 84 browser details YourSeq 68 2185 2685 3000 68.1% chr12 + 55678030 55678276 247 browser details YourSeq 67 1188 1271 3000 90.4% chr7 - 19735750 19735833 84 browser details YourSeq 67 1188 1271 3000 90.4% chr5 - 139517333 139517416 84 browser details YourSeq 67 1191 1271 3000 88.8% chr5 - 140690860 140690939 80 browser details YourSeq 66 1188 1271 3000 87.9% chr5 - 139937078 139937160 83 browser details YourSeq 65 1191 1271 3000 91.2% chr7 - 19436641 19735617 298977 browser details YourSeq 65 1188 1271 3000 84.0% chr5 + 142719350 142719430 81 browser details YourSeq 65 1186 1271 3000 89.3% chr5 + 142849409 142849494 86 browser details YourSeq 65 1188 1271 3000 89.2% chr5 + 140739400 140739483 84 browser details YourSeq 64 1188 1271 3000 89.1% chr7 - 31005853 31062286 56434 browser details YourSeq 64 1191 1271 3000 87.4% chr5 - 143347332 143347411 80 browser details YourSeq 64 1191 1271 3000 84.7% chr5 - 142393844 142393921 78 browser details YourSeq 64 1188 1269 3000 87.5% chr5 - 140920155 140920235 81 browser details YourSeq 64 1186 1271 3000 88.3% chr7 + 35631904 35631991 88 browser details YourSeq 64 1191 1271 3000 90.0% chr7 + 34569362 34569442 81 browser details YourSeq 64 1191 1271 3000 90.0% chr7 + 29065876 29065956 81

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% chr5 + 135783725 135786724 3000 browser details YourSeq 221 1274 1901 3000 87.1% chr5 + 135422734 135423177 444 browser details YourSeq 183 1336 2036 3000 78.6% chr8 + 40523163 40523626 464 browser details YourSeq 171 458 2632 3000 79.2% chr4 + 39823623 39823893 271 browser details YourSeq 154 1088 1816 3000 85.1% chr5 + 140587292 140588000 709 browser details YourSeq 152 1318 1968 3000 81.2% chr8 + 95631422 95631909 488 browser details YourSeq 150 1623 2015 3000 85.8% chrX + 73744862 73745272 411 browser details YourSeq 144 1434 1924 3000 80.1% chr7 + 45311471 45311978 508 browser details YourSeq 138 1774 2004 3000 85.3% chr9 + 94451906 94452159 254 browser details YourSeq 137 1433 2035 3000 79.4% chr4 + 116654668 116655165 498 browser details YourSeq 130 1318 2005 3000 78.0% chr1 + 125481625 125482145 521 browser details YourSeq 109 1729 1932 3000 87.3% chr8 - 94072985 94073234 250 browser details YourSeq 109 1769 2036 3000 88.2% chr18 + 67525175 67525479 305 browser details YourSeq 106 1315 2007 3000 78.1% chrX - 134591229 134591702 474 browser details YourSeq 101 1865 2036 3000 82.0% chr5 + 31325279 31325487 209 browser details YourSeq 99 951 1522 3000 89.0% chr4 - 138165227 138212991 47765 browser details YourSeq 97 1424 1569 3000 84.6% chr11 + 70735071 70735210 140 browser details YourSeq 94 2512 2635 3000 89.3% chr4 - 36003679 36003800 122 browser details YourSeq 92 1274 1820 3000 70.6% chr16 + 11536167 11536531 365 browser details YourSeq 92 1424 1553 3000 86.0% chr12 + 89133981 89134106 126

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

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Gene and protein information: Mdh2 malate dehydrogenase 2, NAD (mitochondrial) [ Mus musculus (house mouse) ] Gene ID: 17448, updated on 12-Aug-2019

Gene summary

Official Symbol Mdh2 provided by MGI Official Full Name malate dehydrogenase 2, NAD (mitochondrial) provided by MGI Primary source MGI:MGI:97050 See related Ensembl:ENSMUSG00000019179 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as MDH; Mor1; Mdh-2; Mor-1 Expression Broad expression in duodenum adult (RPKM 1627.0), heart adult (RPKM 883.1) and 27 other tissues See more Orthologs human all

Genomic context

Location: 5 G2; 5 75.4 cM See Mdh2 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (135778649..135790386)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (136254519..136266256)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 7 transcripts

Gene: Mdh2 ENSMUSG00000019179

Description malate dehydrogenase 2, NAD (mitochondrial) [Source:MGI Symbol;Acc:MGI:97050] Gene Synonyms Mdh-2, Mor-1, Mor1 Location Chromosome 5: 135,778,480-135,790,398 forward strand. GRCm38:CM000998.2 About this gene This gene has 7 transcripts (splice variants), 230 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mdh2- ENSMUST00000019323.10 1456 338aa ENSMUSP00000019323.6 Protein coding CCDS19746 P08249 TSL:1 201 GENCODE basic APPRIS P1

Mdh2- ENSMUST00000200556.4 648 199aa ENSMUSP00000142993.1 Protein coding - A0A0G2JF23 CDS 3' 207 incomplete TSL:3

Mdh2- ENSMUST00000196285.1 506 153aa ENSMUSP00000143748.1 Protein coding - A0A0G2JGY4 CDS 3' 205 incomplete TSL:3

Mdh2- ENSMUST00000138101.1 1112 51aa ENSMUSP00000136225.1 Nonsense mediated - J3QMC8 TSL:5 203 decay

Mdh2- ENSMUST00000197909.1 1606 No - Retained intron - - TSL:NA 206 protein

Mdh2- ENSMUST00000130795.1 725 No - Retained intron - - TSL:2 202 protein

Mdh2- ENSMUST00000143747.1 528 No - Retained intron - - TSL:2 204 protein

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31.92 kb Forward strand

135.77Mb 135.78Mb 135.79Mb 135.80Mb Genes (Comprehensive set... Mdh2-201 >protein coding

Mdh2-206 >retained intron

Mdh2-203 >nonsense mediated decay

Mdh2-202 >retained intron

Mdh2-207 >protein coding

Mdh2-205 >protein coding

Mdh2-204 >retained intron

Contigs AC083948.3 > Genes < Styxl1-201protein coding (Comprehensive set...

< Styxl1-208protein coding

< Styxl1-207protein coding

< Styxl1-204protein coding

< Styxl1-205protein coding

< Styxl1-202protein coding

< Styxl1-206nonsense mediated decay

< Styxl1-203protein coding

< Styxl1-209protein coding

Regulatory Build

135.77Mb 135.78Mb 135.79Mb 135.80Mb Reverse strand 31.92 kb

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

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000019323

11.91 kb Forward strand

Mdh2-201 >protein coding

ENSMUSP00000019... Low complexity (Seg) TIGRFAM Malate dehydrogenase, type 1 Superfamily NAD(P)-binding domain superfamily Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal

Pfam Lactate/malate dehydrogenase, N-terminal Lactate/malate dehydrogenase, C-terminal

PROSITE patterns Malate dehydrogenase, active site PIRSF L-lactate/malate dehydrogenase PANTHER PTHR11540

PTHR11540:SF51 Gene3D 3.40.50.720 Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal

CDD cd01337

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 338

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

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