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Mouse Ndufa4l2 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ndufa4l2 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ndufa4l2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ndufa4l2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ndufa4l2 gene (NCBI Reference Sequence: NM_001098789 ; Ensembl: ENSMUSG00000040280 ) is located on Mouse chromosome 10. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000035735). 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 Ndufa4l2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-327N14 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 22.22% 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: 360 bp, and the size of intron 2 for 3'-loxP site insertion: 777 bp. The size of effective cKO region: ~519 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 4 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Ndufa4l2 cKO region Exon of mouse Shmt2 loxP site Page 2 of 8 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(6949bp) | A(22.85% 1588) | C(28.36% 1971) | T(20.18% 1402) | G(28.61% 1988) 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 8 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 + 127512220 127515219 3000 browser details YourSeq 38 245 282 3000 100.0% chrX + 44576095 44576132 38 browser details YourSeq 26 2871 2907 3000 96.5% chr16 - 84567296 84567334 39 browser details YourSeq 23 2165 2187 3000 100.0% chr9 - 69579806 69579828 23 browser details YourSeq 22 1426 1447 3000 100.0% chrX - 8264080 8264101 22 browser details YourSeq 22 1426 1447 3000 100.0% chr1 + 49496671 49496692 22 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 + 127515739 127518738 3000 browser details YourSeq 800 1535 2834 3000 90.3% chr7 - 80430204 80431261 1058 browser details YourSeq 51 525 591 3000 88.1% chr6 - 11905198 11905264 67 browser details YourSeq 33 1712 1752 3000 90.3% chr5 + 23766140 23766180 41 browser details YourSeq 29 1742 1820 3000 96.8% chr13 - 108266791 108266870 80 browser details YourSeq 26 1791 1820 3000 96.6% chr11 - 6423835 6423869 35 browser details YourSeq 23 1796 1820 3000 87.5% chr12 - 22534778 22534801 24 browser details YourSeq 23 1796 1820 3000 87.5% chr12 - 21726073 21726096 24 browser details YourSeq 23 1796 1820 3000 87.5% chr12 + 19435926 19435949 24 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Ndufa4l2 Ndufa4, mitochondrial complex associated like 2 [ Mus musculus (house mouse) ] Gene ID: 407790, updated on 12-Aug-2019 Gene summary Official Symbol Ndufa4l2 provided by MGI Official Full Name Ndufa4, mitochondrial complex associated like 2 provided by MGI Primary source MGI:MGI:3039567 See related Ensembl:ENSMUSG00000040280 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 BC064011 Expression Broad expression in lung adult (RPKM 37.3), adrenal adult (RPKM 32.7) and 20 other tissues See more Orthologs human all Genomic context Location: 10; 10 D3 See Ndufa4l2 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (127509274..127517154) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (126951995..126954210) Chromosome 10 - NC_000076.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Ndufa4l2 ENSMUSG00000040280 Description Ndufa4, mitochondrial complex associated like 2 [Source:MGI Symbol;Acc:MGI:3039567] Location Chromosome 10: 127,514,967-127,517,154 forward strand. GRCm38:CM001003.2 About this gene This gene has 3 transcripts (splice variants), 198 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ndufa4l2-201 ENSMUST00000035735.10 934 87aa ENSMUSP00000042185.9 Protein coding CCDS36080 Q4FZG9 TSL:1 GENCODE basic APPRIS P1 Ndufa4l2-202 ENSMUST00000218302.1 994 No protein - lncRNA - - TSL:3 Ndufa4l2-203 ENSMUST00000218492.1 377 No protein - lncRNA - - TSL:2 Page 6 of 8 https://www.alphaknockout.com 22.19 kb Forward strand 127.505Mb 127.510Mb 127.515Mb 127.520Mb 127.525Mb Genes (Comprehensive set... Stac3-202 >protein coding Ndufa4l2-201 >protein coding Stac3-201 >protein coding Ndufa4l2-202 >lncRNA Stac3-204 >retained intron Ndufa4l2-203 >lncRNA Stac3-205 >retained intron Contigs AC167719.2 > Genes < Shmt2-206protein coding < Nxph4-201protein coding (Comprehensive set... < Shmt2-201protein coding < Shmt2-207retained intron < Shmt2-203retained intron < Shmt2-204lncRNA < Shmt2-205lncRNA < Shmt2-202retained intron Regulatory Build 127.505Mb 127.510Mb 127.515Mb 127.520Mb 127.525Mb Reverse strand 22.19 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000035735 2.19 kb Forward strand Ndufa4l2-201 >protein coding ENSMUSP00000042... Transmembrane heli... Pfam NADH-ubiquinone reductase complex 1 MLRQ subunit PANTHER PTHR14256:SF5 NADH-ubiquinone reductase complex 1 MLRQ subunit All sequence SNPs/i... Sequence variants (dbSNP and all other sources) R Y Variant Legend missense variant synonymous variant Scale bar 0 8 16 24 32 40 48 56 64 72 87 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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