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Mouse Pcyt1b Conditional Knockout Project (CRISPR/Cas9)

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Mouse Pcyt1b Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pcyt1b Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pcyt1b conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pcyt1b gene (NCBI Reference Sequence: NM_211138 ; Ensembl: ENSMUSG00000035246 ) is located on Mouse chromosome X. 8 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 8 (Transcript: ENSMUST00000045898). 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 Pcyt1b gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-102H18 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 reduced fertility, abnormal ovaries with absent corpora lutea and follicles, benign ovarian tumors, seminiferous tubule degeneration, and reduced spermatogenesis. Exon 2 starts from about 10.66% 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: 26608 bp, and the size of intron 2 for 3'-loxP site insertion: 4373 bp. The size of effective cKO region: ~600 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 8 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pcyt1b 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. 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(7100bp) | A(27.52% 1954) | C(22.27% 1581) | T(29.42% 2089) | G(20.79% 1476) 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% chrX + 93698836 93701835 3000 browser details YourSeq 108 465 2800 3000 92.2% chr2 - 71267298 71371465 104168 browser details YourSeq 89 465 647 3000 76.6% chr12 + 103120407 103120605 199 browser details YourSeq 86 460 669 3000 81.2% chr11 - 58104796 58105322 527 browser details YourSeq 85 471 669 3000 76.1% chr4 + 88022687 88023103 417 browser details YourSeq 84 468 651 3000 73.6% chr13 + 42765409 42765575 167 browser details YourSeq 82 462 663 3000 71.0% chr2 + 91369509 91369711 203 browser details YourSeq 81 431 596 3000 84.5% chr7 + 26319916 26320162 247 browser details YourSeq 78 271 597 3000 67.7% chr19 - 14427799 14427969 171 browser details YourSeq 73 431 596 3000 86.9% chr7 + 26957450 26957696 247 browser details YourSeq 71 460 598 3000 91.0% chr18 - 61062630 61063074 445 browser details YourSeq 70 465 610 3000 84.2% chr4 - 42346735 42346874 140 browser details YourSeq 70 465 610 3000 84.2% chr4 - 41994520 41994659 140 browser details YourSeq 70 463 653 3000 71.5% chr2 - 107698388 107698542 155 browser details YourSeq 68 465 610 3000 83.8% chr4 - 41836257 41836396 140 browser details YourSeq 68 466 601 3000 78.8% chr2 - 74871799 74871932 134 browser details YourSeq 68 449 596 3000 80.7% chr7 + 26848208 26848351 144 browser details YourSeq 67 460 572 3000 79.7% chr17 + 72315384 72315496 113 browser details YourSeq 66 468 581 3000 79.0% chr2 - 35419652 35419765 114 browser details YourSeq 65 465 610 3000 72.5% chr2 - 161815002 161815150 149 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% chrX + 93702436 93705435 3000 browser details YourSeq 64 2619 2702 3000 91.1% chr4 + 120753894 120754003 110 browser details YourSeq 63 2633 2712 3000 94.5% chr5 - 50890472 50890814 343 browser details YourSeq 59 2619 2701 3000 91.0% chr11 - 120034635 120034727 93 browser details YourSeq 56 2435 2712 3000 68.8% chr11 - 105676225 105676328 104 browser details YourSeq 56 2617 2705 3000 81.5% chr11 + 78387603 78387684 82 browser details YourSeq 54 2636 2712 3000 95.1% chr3 - 69695366 69695469 104 browser details YourSeq 51 2628 2713 3000 81.7% chr6 + 96338316 96338395 80 browser details YourSeq 51 2628 2690 3000 94.9% chr1 + 129166620 129166716 97 browser details YourSeq 48 2655 2711 3000 94.5% chr14 - 58436816 58436874 59 browser details YourSeq 46 2646 2706 3000 81.2% chr10 + 119949209 119949263 55 browser details YourSeq 46 2626 2693 3000 96.2% chr1 + 141475146 141475218 73 browser details YourSeq 44 2640 2706 3000 92.6% chr14 + 73065574 73065662 89 browser details YourSeq 42 2643 2704 3000 88.7% chr1 + 86623817 86623883 67 browser details YourSeq 41 2624 2688 3000 93.8% chr12 - 10854929 10855003 75 browser details YourSeq 40 2662 2705 3000 97.7% chr1 + 154072353 154072429 77 browser details YourSeq 39 2629 2684 3000 91.4% chr10 - 92383286 92383341 56 browser details YourSeq 39 2626 2674 3000 93.4% chr3 + 151499579 151499641 63 browser details YourSeq 39 2619 2676 3000 74.5% chr13 + 54487063 54487105 43 browser details YourSeq 39 2634 2674 3000 100.0% chr10 + 121070110 121070320 211 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: Pcyt1b phosphate cytidylyltransferase 1, choline, beta isoform [ Mus musculus (house mouse) ] Gene ID: 236899, updated on 12-Aug-2019 Gene summary Official Symbol Pcyt1b provided by MGI Official Full Name phosphate cytidylyltransferase 1, choline, beta isoform provided by MGI Primary source MGI:MGI:2147987 See related Ensembl:ENSMUSG00000035246 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 CTTbeta; AW045697 Expression Biased expression in CNS E18 (RPKM 9.3), CNS E14 (RPKM 7.0) and 12 other tissues See more Orthologs human all Genomic context Location: X; X C3 See Pcyt1b in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (93654863..93749951) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (90900202..90995287) Chromosome X - NC_000086.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Pcyt1b ENSMUSG00000035246 Description phosphate cytidylyltransferase 1, choline, beta isoform [Source:MGI Symbol;Acc:MGI:2147987] Gene Synonyms CTTbeta Location Chromosome X: 93,654,863-93,749,951 forward strand. GRCm38:CM001013.2 About this gene This gene has 3 transcripts (splice variants), 259 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pcyt1b-201 ENSMUST00000045898.3 4888 369aa ENSMUSP00000044280.3 Protein coding CCDS30274 Q811Q9 TSL:1 GENCODE basic APPRIS P1 Pcyt1b-202 ENSMUST00000113933.8 4867 339aa ENSMUSP00000109566.2 Protein coding CCDS41061 Q811Q9 TSL:1 GENCODE basic Pcyt1b-203 ENSMUST00000146263.1 350 No protein - lncRNA - - TSL:3 115.09 kb Forward strand 93.66Mb 93.68Mb 93.70Mb 93.72Mb 93.74Mb Genes (Comprehensive set... Pcyt1b-202 >protein coding Pcyt1b-201 >protein coding Pcyt1b-203 >lncRNA Contigs AL589652.13 > Regulatory Build 93.66Mb 93.68Mb 93.70Mb 93.72Mb 93.74Mb Reverse strand 115.09 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000045898 74.85 kb Forward strand Pcyt1b-201 >protein coding ENSMUSP00000044... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) TIGRFAM Cytidyltransferase-like domain Superfamily SSF52374 Pfam Cytidyltransferase-like domain PANTHER PTHR10739:SF20 PTHR10739 Gene3D Rossmann-like alpha/beta/alpha sandwich fold CDD CTP:phosphocholine cytidylyltransferase domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 369 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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