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Mouse Pkib Conditional Knockout Project (CRISPR/Cas9)

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Mouse Pkib Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pkib Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pkib conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pkib gene (NCBI Reference Sequence: NM_008863 ; Ensembl: ENSMUSG00000019876 ) is located on Mouse chromosome 10. 6 exons are identified, with the ATG start codon in exon 4 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000095668). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pkib gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-338H6 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: Homozygotes for a targeted null mutation exhibit reduced protein kinase inhibitor activity in the testis but are phenotypically normal and fertile. Exon 5 is not frameshift exon, and covers 53.64% of the coding region. The size of intron 4 for 5'-loxP site insertion: 19908 bp, and the size of intron 5 for 3'-loxP site insertion: 8023 bp. The size of effective cKO region: ~677 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 5 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pkib Homology arm cKO region 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. 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(7177bp) | A(29.43% 2112) | C(20.29% 1456) | T(28.93% 2076) | G(21.36% 1533) 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 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 + 57724841 57727840 3000 browser details YourSeq 192 1579 2349 3000 82.6% chr17 + 48618411 48619296 886 browser details YourSeq 146 2070 2365 3000 87.4% chr14 - 68165426 68165898 473 browser details YourSeq 139 1508 2121 3000 91.8% chr18 - 12090549 12091182 634 browser details YourSeq 135 1943 2360 3000 90.0% chr11 - 49569742 49570352 611 browser details YourSeq 134 2123 2366 3000 82.0% chr2 + 130087246 130087805 560 browser details YourSeq 133 1911 2349 3000 85.9% chr19 - 22234040 22234652 613 browser details YourSeq 132 1753 2361 3000 72.8% chr9 - 70147262 70147888 627 browser details YourSeq 131 1773 2351 3000 78.3% chr15 - 94679785 94680450 666 browser details YourSeq 125 1700 2350 3000 87.4% chrX - 135444003 135505363 61361 browser details YourSeq 124 2111 2343 3000 90.8% chr3 - 85958954 85959587 634 browser details YourSeq 123 1775 2367 3000 89.2% chr13 - 30769217 30769882 666 browser details YourSeq 122 1903 2340 3000 90.2% chr3 + 105325569 105326105 537 browser details YourSeq 121 2009 2364 3000 88.1% chr14 + 47614850 47615455 606 browser details YourSeq 120 2086 2358 3000 92.4% chr5 - 147197625 147198324 700 browser details YourSeq 120 2070 2352 3000 88.4% chr16 - 87760197 87760684 488 browser details YourSeq 120 1943 2340 3000 90.6% chr11 - 51156194 51156790 597 browser details YourSeq 120 1909 2352 3000 88.9% chr2 + 147166006 147166671 666 browser details YourSeq 116 1699 2341 3000 88.6% chr14 - 35736931 35737575 645 browser details YourSeq 115 1520 2088 3000 89.2% chrX - 142484107 142484697 591 Note: The 3000 bp section upstream of Exon 5 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 + 57728518 57731517 3000 browser details YourSeq 395 512 1285 3000 87.2% chr8 - 89506298 89507096 799 browser details YourSeq 393 552 1285 3000 82.7% chr13 - 57334259 57335006 748 browser details YourSeq 390 539 1285 3000 88.3% chr4 - 52209023 52209810 788 browser details YourSeq 380 526 1284 3000 81.0% chr18 - 82450817 82451641 825 browser details YourSeq 375 556 1150 3000 86.4% chr13 - 67495769 67496375 607 browser details YourSeq 375 570 1276 3000 82.5% chr1 + 34491252 34491947 696 browser details YourSeq 367 605 1285 3000 82.2% chr7 + 118256864 118257549 686 browser details YourSeq 366 524 1285 3000 88.7% chr15 + 90857045 90857819 775 browser details YourSeq 365 524 1204 3000 86.8% chr4 + 74238803 74239515 713 browser details YourSeq 364 505 1145 3000 88.6% chr3 - 96588834 96589663 830 browser details YourSeq 363 553 1177 3000 86.9% chr7 - 130285609 130286311 703 browser details YourSeq 358 622 1284 3000 87.0% chr8 - 77334070 77334721 652 browser details YourSeq 357 572 1115 3000 87.1% chr5 + 148455301 148455835 535 browser details YourSeq 357 541 1204 3000 85.4% chr4 + 48379312 48379944 633 browser details YourSeq 353 524 1127 3000 88.7% chr8 + 120815970 120816589 620 browser details YourSeq 347 556 1101 3000 88.4% chr17 - 47041572 47042156 585 browser details YourSeq 347 556 1142 3000 89.2% chr14 + 20132318 20132931 614 browser details YourSeq 345 571 1204 3000 86.0% chr10 - 88395891 88396671 781 browser details YourSeq 343 585 1283 3000 88.6% chr5 + 20204356 20205058 703 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Pkib protein kinase inhibitor beta, cAMP dependent, testis specific [ Mus musculus (house mouse) ] Gene ID: 18768, updated on 12-Aug-2019 Gene summary Official Symbol Pkib provided by MGI Official Full Name protein kinase inhibitor beta, cAMP dependent, testis specific provided by MGI Primary source MGI:MGI:101937 See related Ensembl:ENSMUSG00000019876 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 Pkibeta; Prkacn2 Expression Biased expression in testis adult (RPKM 8.2), cerebellum adult (RPKM 3.8) and 3 other tissues See more Orthologs human all Genomic context Location: 10 B4; 10 29.22 cM See Pkib in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (57631981..57741112) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (57351787..57460918) Chromosome 10 - NC_000076.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 11 transcripts Gene: Pkib ENSMUSG00000019876 Description protein kinase inhibitor beta, cAMP dependent, testis specific [Source:MGI Symbol;Acc:MGI:101937] Gene Synonyms PKIbeta, Prkacn2 Location Chromosome 10: 57,631,981-57,741,112 forward strand. GRCm38:CM001003.2 About this gene This gene has 11 transcripts (splice variants), 116 orthologues, 2 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 Pkib-202 ENSMUST00000075992.11 5317 78aa ENSMUSP00000075374.5 Protein coding CCDS23855 Q3TZU5 TSL:1 GENCODE basic APPRIS P1 Pkib-203 ENSMUST00000095668.9 3311 110aa ENSMUSP00000093329.3 Protein coding CCDS23854 F7D7A9 TSL:1 GENCODE basic Pkib-201 ENSMUST00000066028.12 1552 78aa ENSMUSP00000067532.6 Protein coding CCDS23855 Q3TZU5 TSL:1 GENCODE basic APPRIS P1 Pkib-204 ENSMUST00000175852.1 731 78aa ENSMUSP00000135666.1 Protein coding CCDS23855 Q3TZU5 TSL:1 GENCODE basic APPRIS P1 Pkib-209 ENSMUST00000177325.7 540 78aa ENSMUSP00000135280.1 Protein coding CCDS23855 Q3TZU5 TSL:1 GENCODE basic APPRIS P1 Pkib-211 ENSMUST00000177473.7 525 78aa ENSMUSP00000135835.1 Protein coding CCDS23855 Q3TZU5 TSL:3 GENCODE basic APPRIS P1 Pkib-210 ENSMUST00000177438.7 417 24aa ENSMUSP00000135774.1 Protein coding - H3BLG2 CDS 3' incomplete TSL:3 Pkib-205 ENSMUST00000176225.1 2773 No protein - Retained intron - - TSL:1 Pkib-208 ENSMUST00000177299.7 2608 No protein - Retained intron - - TSL:1 Pkib-207 ENSMUST00000176741.7 2528 No protein - lncRNA - - TSL:2 Pkib-206 ENSMUST00000176354.1 324 No protein - lncRNA - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 129.13 kb Forward strand 57.64Mb 57.66Mb 57.68Mb 57.70Mb 57.72Mb 57.74Mb Genes (Comprehensive set... Pkib-203 >protein coding Pkib-202 >protein coding Pkib-208 >retained intron Pkib-206 >lncRNA Pkib-205 >retained intron Pkib-210 >protein coding Pkib-207 >lncRNA Pkib-209 >protein coding Pkib-211 >protein coding Pkib-201 >protein coding Pkib-204 >protein coding Contigs AC102647.8 > AC168055.3 > Regulatory Build 57.64Mb 57.66Mb 57.68Mb 57.70Mb 57.72Mb 57.74Mb Reverse strand 129.13 kb Regulation Legend
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