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Mouse Lim2 Conditional Knockout Project (CRISPR/Cas9)

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Mouse Lim2 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Lim2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Lim2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Lim2 gene (NCBI Reference Sequence: NM_177693 ; Ensembl: ENSMUSG00000093639 ) is located on Mouse chromosome 7. 5 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000004732). 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 Lim2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-77K23 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 mutants exhibit microphthalmia, total lens opacity, disorganized lens fibers, posterior rupturing of lens capsule, and some have vacuolated lens. Heterozygotes exhibit the same dense cataract, but without microphthalmia or lens rupture. Exon 2 starts from about 0.39% 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: 428 bp, and the size of intron 2 for 3'-loxP site insertion: 2802 bp. The size of effective cKO region: ~639 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 gRNA region 5' gRNA region 3' 1 2 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Lim2 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7103bp) | A(27.43% 1948) | C(24.62% 1749) | T(24.07% 1710) | G(23.88% 1696) 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% chr7 + 43427378 43430377 3000 browser details YourSeq 161 528 752 3000 98.3% chr2 + 120165793 120166280 488 browser details YourSeq 154 198 679 3000 93.5% chr1 - 166193134 166193610 477 browser details YourSeq 153 459 679 3000 97.0% chr1 - 152802460 152802756 297 browser details YourSeq 150 528 752 3000 88.9% chrX + 13016305 13016475 171 browser details YourSeq 150 516 678 3000 96.4% chr17 + 29657447 29657610 164 browser details YourSeq 149 527 679 3000 98.7% chr5 + 137593513 137593665 153 browser details YourSeq 148 527 679 3000 98.7% chr17 - 3569376 3569529 154 browser details YourSeq 147 527 679 3000 98.1% chr4 - 55450263 55450415 153 browser details YourSeq 147 525 679 3000 97.5% chr3 - 62372131 62372285 155 browser details YourSeq 147 523 678 3000 97.5% chr4 + 133760465 133760621 157 browser details YourSeq 146 524 679 3000 97.5% chr8 - 111353171 111353332 162 browser details YourSeq 146 527 679 3000 98.1% chr12 - 113054380 113054533 154 browser details YourSeq 146 527 679 3000 98.1% chr2 + 76985529 76985682 154 browser details YourSeq 146 526 679 3000 98.1% chr12 + 86457995 86458151 157 browser details YourSeq 145 528 679 3000 98.1% chr9 - 53360855 53361007 153 browser details YourSeq 145 529 681 3000 96.1% chr17 - 85219810 85219961 152 browser details YourSeq 145 527 679 3000 97.4% chr17 - 29987581 29987733 153 browser details YourSeq 145 529 679 3000 96.7% chr13 - 19994672 19994821 150 browser details YourSeq 145 527 679 3000 97.4% chr10 - 4345102 4345254 153 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% chr7 + 43431017 43434016 3000 browser details YourSeq 115 1249 1381 3000 95.4% chr4 - 38546843 38547403 561 browser details YourSeq 110 1262 1380 3000 97.5% chr8 + 110511150 110511698 549 browser details YourSeq 107 1263 1380 3000 95.8% chr7 - 17577705 17578224 520 browser details YourSeq 103 714 1008 3000 88.9% chr8 + 80617092 80617390 299 browser details YourSeq 98 1262 1380 3000 96.3% chr12 + 18362126 18362675 550 browser details YourSeq 88 888 1006 3000 90.1% chrX + 162449686 162449804 119 browser details YourSeq 82 1296 1382 3000 97.8% chr2 - 26801671 26801773 103 browser details YourSeq 81 1299 1380 3000 100.0% chr2 + 4925422 4925528 107 browser details YourSeq 79 1300 1380 3000 98.8% chr7 - 17569676 17569756 81 browser details YourSeq 79 1296 1380 3000 96.5% chr7 - 17496767 17496851 85 browser details YourSeq 79 1300 1380 3000 98.8% chr7 - 17488720 17488800 81 browser details YourSeq 79 1300 1380 3000 98.8% chr7 - 17351982 17352062 81 browser details YourSeq 79 801 1011 3000 88.5% chr10 + 112205692 112205921 230 browser details YourSeq 79 1262 1362 3000 96.6% chr10 + 83743336 83743870 535 browser details YourSeq 78 1299 1380 3000 98.8% chr12 - 24385797 24385903 107 browser details YourSeq 75 710 1001 3000 89.1% chr5 + 148445531 148445821 291 browser details YourSeq 74 1302 1379 3000 97.5% chr7 - 17359969 17360046 78 browser details YourSeq 72 1302 1379 3000 96.2% chr7 - 17420266 17420343 78 browser details YourSeq 72 1302 1379 3000 96.2% chr7 - 17311082 17311159 78 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: Lim2 lens intrinsic membrane protein 2 [ Mus musculus (house mouse) ] Gene ID: 233187, updated on 8-Oct-2019 Gene summary Official Symbol Lim2 provided by MGI Official Full Name lens intrinsic membrane protein 2 provided by MGI Primary source MGI:MGI:104698 See related Ensembl:ENSMUSG00000118560 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 To3; MP17; MP18; MP19; MP20; 4833403J20 Expression Low expression observed in reference dataset See more Orthologs human all Genomic context Location: 7 B3; 7 28.25 cM See Lim2 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (43430099..43435996) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (50685471..50691361) Chromosome 7 - NC_000073.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Lim2 ENSMUSG00000093639 Description lens intrinsic membrane protein 2 [Source:MGI Symbol;Acc:MGI:104698] Gene Synonyms 19kDa, MP19 Location Chromosome 7: 43,427,670-43,435,991 forward strand. GRCm38:CM001000.2 About this gene This gene has 1 transcript (splice variant), 60 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Lim2-201 ENSMUST00000177375.1 2180 185aa ENSMUSP00000158870.1 Nonsense mediated decay - H3BKT1 TSL:1 GENCODE basic APPRIS P1 28.32 kb Forward strand 43.42Mb 43.43Mb 43.44Mb Genes (Comprehensive set... Siglecg-201 >protein coding Lim2-201 >nonsense mediated decay Nkg7-201 >protein codinCgldnd2-201 >protein coding Siglecg-206 >retained intron AC149091.1-201 >protein coding Nkg7-203 >protein coding Cldnd2-202 >protein coding Siglecg-203 >retained intron Lim2-202 >protein coding Nkg7-202 >retained intron Etfb-201 >protein coding Etfbl-201 >nonsense mediated decay Etfbl-202 >nonsense mediated decay Etfb-206 >protein coding Etfb-205 >protein coding Etfb-207 >protein coding Contigs AC149091.3 > < AC154108.3 Regulatory Build 43.42Mb 43.43Mb 43.44Mb Reverse strand 28.32 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000004732 8.32 kb Forward strand Lim2-201 >nonsense mediated decay ENSMUSP00000158... All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 185 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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