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Mouse Ldhb Conditional Knockout Project (CRISPR/Cas9)

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Mouse Ldhb Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ldhb Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ldhb conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ldhb gene (NCBI Reference Sequence: NM_008492 ; Ensembl: ENSMUSG00000030246 ) is located on Mouse chromosome 6. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000032373). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ldhb gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-134H20 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: Electrophoretic variants of LDHB are determined by: the a allele with fast anodal mobility in all inbred strains tested; and the b allele with slower mobility in Peru-Coppock stock. Three additional variants are known in wild M. spretus from southern France and Spain. Alleles are codominant. Exon 3 starts from about 12.97% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 3954 bp, and the size of intron 3 for 3'-loxP site insertion: 2661 bp. The size of effective cKO region: ~1353 bp. The transcript Ldhb-202 may not be affected by deleting this cKO region. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 3 8 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ldhb Homology arm 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. 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(7118bp) | A(25.25% 1797) | C(22.3% 1587) | T(30.46% 2168) | G(22.0% 1566) 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. 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% chr6 - 142501708 142504707 3000 browser details YourSeq 69 802 970 3000 73.4% chr12 - 51731183 51731323 141 browser details YourSeq 66 702 963 3000 82.4% chr6 - 136432809 136433087 279 browser details YourSeq 51 79 166 3000 78.0% chr14 - 20116855 20116926 72 browser details YourSeq 49 79 159 3000 77.6% chr19 + 9994064 9994128 65 browser details YourSeq 44 892 984 3000 87.3% chr11 - 4144867 4144958 92 browser details YourSeq 42 805 963 3000 74.5% chr13 - 15489033 15489191 159 browser details YourSeq 42 804 955 3000 76.8% chr11 - 79174217 79174361 145 browser details YourSeq 40 81 159 3000 71.2% chr11 + 89453425 89453477 53 browser details YourSeq 39 116 163 3000 84.8% chr12 - 101646984 101647029 46 browser details YourSeq 39 120 163 3000 85.0% chr11 + 8955514 8955553 40 browser details YourSeq 38 120 163 3000 95.3% chr11 - 116693406 116693452 47 browser details YourSeq 37 874 970 3000 93.2% chr10 + 64145184 64145282 99 browser details YourSeq 35 121 160 3000 94.9% chr13 - 89712750 89712791 42 browser details YourSeq 35 122 162 3000 92.7% chr12 - 83652016 83652056 41 browser details YourSeq 35 1712 1753 3000 92.7% chr11 + 118314264 118314305 42 browser details YourSeq 34 577 621 3000 89.5% chr18 - 63687406 63687449 44 browser details YourSeq 34 121 163 3000 94.9% chr10 - 102517829 102517871 43 browser details YourSeq 34 113 162 3000 72.3% chr1 - 50071519 50071554 36 browser details YourSeq 33 574 624 3000 92.5% chr13 - 99095618 99095670 53 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 - 142498090 142501089 3000 browser details YourSeq 199 45 481 3000 86.5% chr10 - 76083891 76084186 296 browser details YourSeq 198 45 484 3000 86.5% chr10 - 85083324 85083566 243 browser details YourSeq 193 96 482 3000 96.7% chr13 - 49573460 49573906 447 browser details YourSeq 184 286 481 3000 95.3% chr7 - 122093309 122093499 191 browser details YourSeq 184 45 481 3000 86.2% chr15 - 97427699 97427956 258 browser details YourSeq 184 45 481 3000 87.0% chr16 + 4893269 4893526 258 browser details YourSeq 183 96 481 3000 95.6% chr8 - 33969228 33969781 554 browser details YourSeq 182 97 481 3000 87.7% chr15 + 99821982 99822205 224 browser details YourSeq 181 96 481 3000 87.0% chrX - 101583358 101583558 201 browser details YourSeq 181 97 481 3000 89.2% chr7 + 64965871 64966091 221 browser details YourSeq 179 262 498 3000 87.8% chr4 - 136333696 136333914 219 browser details YourSeq 179 100 481 3000 89.4% chr2 - 29911060 29911262 203 browser details YourSeq 178 96 481 3000 89.0% chr7 - 6975919 6976139 221 browser details YourSeq 178 285 481 3000 94.3% chr17 - 16903623 16903816 194 browser details YourSeq 177 96 481 3000 87.5% chr5_JH584296_random - 49452 49647 196 browser details YourSeq 177 97 482 3000 88.0% chr4 - 135256258 135256450 193 browser details YourSeq 177 286 497 3000 91.4% chr18 + 35429564 35429766 203 browser details YourSeq 177 97 482 3000 88.0% chr11 + 120413873 120414065 193 browser details YourSeq 176 286 496 3000 92.4% chr16 - 90174979 90175184 206 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ldhb lactate dehydrogenase B [ Mus musculus (house mouse) ] Gene ID: 16832, updated on 12-Aug-2019 Gene summary Official Symbol Ldhb provided by MGI Official Full Name lactate dehydrogenase B provided by MGI Primary source MGI:MGI:96763 See related Ensembl:ENSMUSG00000030246 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Ldh2; H-Ldh; LDH-B; LDH-H; Ldh-2; AI790582 Summary This gene encodes the B subunit of lactate dehydrogenase enzyme, which catalyzes the interconversion of pyruvate and Expression lactate with concomitant interconversion of NADH and NAD+ in a post-glycolysis process. Alternatively spliced transcript variants have also been found for this gene. Recent studies have shown that a C-terminally extended isoform is produced by use of an alternative in-frame translation termination codon via a stop codon readthrough mechanism, and that this isoform is localized in the peroxisomes. Pseudogenes have been identified on chromosomes 1 and 19. [provided by RefSeq, Feb 2016] Orthologs Broad expression in kidney adult (RPKM 644.8), heart adult (RPKM 618.4) and 18 other tissues See more human all Genomic context Location: 6 G2; 6 74.17 cM See Ldhb in Genome Data Viewer Exon count: 8 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (142490249..142507957, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (142438769..142456463, complement) Chromosome 6 - NC_000072.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Ldhb ENSMUSG00000030246 Description lactate dehydrogenase B [Source:MGI Symbol;Acc:MGI:96763] Gene Synonyms H-Ldh, Ldh-2, lactate dehydrogenase-B Location Chromosome 6: 142,490,249-142,507,957 reverse strand. GRCm38:CM000999.2 About this gene This gene has 4 transcripts (splice variants), 363 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 8 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ldhb-201 ENSMUST00000032373.11 1304 334aa ENSMUSP00000032373.5 Protein coding CCDS20684 P16125 TSL:1 GENCODE basic APPRIS P1 Ldhb-203 ENSMUST00000134191.2 867 198aa ENSMUSP00000116014.1 Protein coding - D3Z7F0 CDS 3' incomplete TSL:2 Ldhb-204 ENSMUST00000204433.2 783 193aa ENSMUSP00000145261.1 Protein coding - A0A0N4SVV8 CDS 5' incomplete TSL:3 Ldhb-202 ENSMUST00000130817.2 437 32aa ENSMUSP00000145467.1 Protein coding - A0A0N4SWC9 CDS 5' incomplete TSL:3 37.71 kb Forward strand 142.49Mb 142.50Mb 142.51Mb Contigs AC142413.4 > Genes (Comprehensive set... < Ldhb-201protein coding < Ldhb-202protein coding < Ldhb-204protein coding < Ldhb-203protein coding Regulatory Build 142.49Mb 142.50Mb 142.51Mb Reverse strand 37.71 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000032373 < Ldhb-201protein coding Reverse strand 17.71 kb ENSMUSP00000032... Coiled-coils (Ncoils) TIGRFAM L-lactate dehydrogenase Superfamily NAD(P)-binding domain superfamily Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal Prints L-lactate/malate dehydrogenase Pfam Lactate/malate dehydrogenase, N-terminal Lactate/malate dehydrogenase, C-terminal PROSITE patterns L-lactate dehydrogenase, active site PIRSF L-lactate/malate dehydrogenase PANTHER PTHR43128:SF2 PTHR43128 HAMAP L-lactate dehydrogenase Gene3D 3.40.50.720 Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal CDD cd05293 All sequence SNPs/i..
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