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Mouse Aktip Conditional Knockout Project (CRISPR/Cas9)

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Mouse Aktip Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Aktip Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Aktip conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Aktip gene (NCBI Reference Sequence: NM_001302267 ; Ensembl: ENSMUSG00000031667 ) is located on Mouse chromosome 8. 11 exons are identified, with the ATG start codon in exon 3 and the TAA stop codon in exon 11 (Transcript: ENSMUST00000120213). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Aktip gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-149I24 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: Mice homozygous for a hypomorphic allele exhibit impaired growth, skeletal and skin defects, abnormal heart tissue, and sterility. Exon 4 starts from about 4.91% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 1235 bp, and the size of intron 4 for 3'-loxP site insertion: 2670 bp. The size of effective cKO region: ~706 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 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Aktip 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(7206bp) | A(25.08% 1807) | C(20.9% 1506) | T(30.16% 2173) | G(23.87% 1720) 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% chr8 - 91130056 91133055 3000 browser details YourSeq 242 685 1312 3000 89.1% chr10 - 61966496 61966777 282 browser details YourSeq 241 685 1312 3000 90.6% chr4 - 119532662 119533220 559 browser details YourSeq 240 688 1312 3000 89.6% chr8 - 4185238 4185515 278 browser details YourSeq 236 692 1312 3000 89.6% chr11 - 103757117 103757504 388 browser details YourSeq 235 681 1311 3000 87.7% chr15 - 102125693 102125993 301 browser details YourSeq 235 680 1312 3000 87.4% chr6 + 113575459 113575791 333 browser details YourSeq 235 685 1312 3000 97.2% chr17 + 74540350 74541003 654 browser details YourSeq 233 687 1312 3000 88.5% chr12 - 55773296 55773605 310 browser details YourSeq 214 689 1096 3000 95.8% chr2 - 34723329 34723968 640 browser details YourSeq 209 692 1096 3000 96.1% chr10 - 128171434 128171896 463 browser details YourSeq 208 685 1269 3000 90.6% chr2 - 119206169 119206658 490 browser details YourSeq 206 681 1096 3000 96.0% chr7 - 97828057 97828654 598 browser details YourSeq 204 683 1310 3000 86.7% chr4 + 116828859 116829365 507 browser details YourSeq 201 693 1096 3000 90.3% chr5 - 122929356 122929730 375 browser details YourSeq 200 681 1096 3000 94.3% chr7 - 144869945 144870528 584 browser details YourSeq 197 694 1096 3000 98.1% chr8 - 107033608 107034019 412 browser details YourSeq 197 681 879 3000 99.5% chr1 - 21334984 21335182 199 browser details YourSeq 196 681 879 3000 99.5% chr15 - 35090900 35091099 200 browser details YourSeq 195 680 879 3000 99.0% chr6 - 85359292 85359493 202 Note: The 3000 bp section upstream of Exon 4 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% chr8 - 91126350 91129349 3000 browser details YourSeq 104 1186 1394 3000 90.8% chr2 - 157415501 157415986 486 browser details YourSeq 96 1202 1405 3000 84.7% chr4 - 129510035 129510266 232 browser details YourSeq 96 1153 1399 3000 90.2% chr16 - 35203120 35203671 552 browser details YourSeq 94 1291 1424 3000 90.6% chr12 + 76811472 76834508 23037 browser details YourSeq 91 1270 1398 3000 89.8% chr5 - 115802253 115802416 164 browser details YourSeq 89 1285 1405 3000 87.7% chr3 - 147583186 147583336 151 browser details YourSeq 88 1086 1400 3000 91.7% chr6 + 41280256 41280665 410 browser details YourSeq 87 1071 1400 3000 93.2% chr8 - 67631000 67631355 356 browser details YourSeq 87 1291 1405 3000 88.7% chr7 - 110248254 110248398 145 browser details YourSeq 87 197 1329 3000 91.5% chr9 + 64419302 64719479 300178 browser details YourSeq 86 1071 1326 3000 89.9% chr17 + 44711469 44756823 45355 browser details YourSeq 85 1291 1399 3000 89.9% chr15 - 96988085 96988223 139 browser details YourSeq 85 866 1400 3000 75.0% chr8 + 119418670 119418815 146 browser details YourSeq 85 1291 1399 3000 89.9% chr12 + 45273777 45273914 138 browser details YourSeq 84 1071 1402 3000 85.5% chr17 - 74928550 74928958 409 browser details YourSeq 84 1291 1404 3000 88.3% chr14 - 112495164 112495304 141 browser details YourSeq 83 1291 1399 3000 88.9% chr2 - 127185734 127185871 138 browser details YourSeq 82 1291 1397 3000 89.6% chr6 - 87970676 87970813 138 browser details YourSeq 82 1290 1398 3000 91.8% chr1 - 166091812 166091922 111 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Aktip thymoma viral proto-oncogene 1 interacting protein [ Mus musculus (house mouse) ] Gene ID: 14339, updated on 24-Oct-2019 Gene summary Official Symbol Aktip provided by MGI Official Full Name thymoma viral proto-oncogene 1 interacting protein provided by MGI Primary source MGI:MGI:3693832 See related Ensembl:ENSMUSG00000031667 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 Ft; Fif; Ft1; Fts; AL023020 Expression Ubiquitous expression in cerebellum adult (RPKM 8.7), bladder adult (RPKM 7.7) and 28 other tissues See more Orthologs human all Genomic context Location: 8 C4; 8 44.25 cM See Aktip in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (91111781..91135494, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (93647398..93659395, complement) Chromosome 8 - NC_000074.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 13 transcripts Gene: Aktip ENSMUSG00000031667 Description thymoma viral proto-oncogene 1 interacting protein [Source:MGI Symbol;Acc:MGI:3693832] Gene Synonyms Ft1 Location Chromosome 8: 91,111,784-91,199,976 reverse strand. GRCm38:CM001001.2 About this gene This gene has 13 transcripts (splice variants), 211 orthologues, 25 paralogues, is a member of 1 Ensembl protein family and is associated with 16 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Aktip- ENSMUST00000120213.8 2200 292aa ENSMUSP00000112375.1 Protein coding CCDS22519 Q64362 TSL:1 202 GENCODE basic APPRIS P1 Aktip- ENSMUST00000120349.7 2153 292aa ENSMUSP00000113769.1 Protein coding CCDS22519 Q64362 TSL:1 203 GENCODE basic APPRIS P1 Aktip- ENSMUST00000125257.2 2079 292aa ENSMUSP00000119277.2 Protein coding CCDS22519 D3Z2J4 TSL:1 205 Q64362 GENCODE basic APPRIS P1 Aktip- ENSMUST00000120426.8 1687 258aa ENSMUSP00000113379.2 Protein coding CCDS85582 D3Z632 TSL:1 204 GENCODE basic Aktip- ENSMUST00000109609.8 1171 292aa ENSMUSP00000105238.2 Protein coding CCDS22519 Q64362 TSL:1 201 GENCODE basic APPRIS P1 Aktip- ENSMUST00000209444.1 474 55aa ENSMUSP00000148050.1 Protein coding - A0A1B0GSS3 CDS 3' 208 incomplete TSL:3 Aktip- ENSMUST00000209311.1 391 51aa ENSMUSP00000147841.1 Protein coding - A0A1B0GS90 CDS 3' 207 incomplete TSL:2 Aktip- ENSMUST00000211050.1 738 32aa ENSMUSP00000147768.1 Nonsense mediated - A0A1B0GS26 CDS 5' 211 decay incomplete TSL:3 Aktip- ENSMUST00000211042.1 3533 No - Retained intron - - TSL:NA 210 protein Aktip- ENSMUST00000210426.1 811 No - lncRNA - - TSL:5 209 protein Aktip- ENSMUST00000211576.1 450 No - lncRNA - - TSL:3 212 protein Aktip- ENSMUST00000211618.1 409 No - lncRNA - - TSL:2 213 protein Aktip- ENSMUST00000153201.2 139 No - lncRNA - - TSL:1 206 protein Page 6 of 8 https://www.alphaknockout.com 108.19 kb Forward strand 91.12Mb 91.14Mb 91.16Mb 91.18Mb 91.20Mb Genes Rbl2-201 >protein coding Gm45665-201 >processed pseudogene (Comprehensive set... Rbl2-210 >protein coding Gm8489-201 >processed pseudogene Rbl2-208 >protein coding Gm45666-201 >lncRNA Rbl2-205 >retained
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