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Mouse Gnai3 Knockout Project (CRISPR/Cas9)

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Mouse Gnai3 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Gnai3 Knockout Project (CRISPR/Cas9) Objective: To create a Gnai3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Gnai3 gene (NCBI Reference Sequence: NM_010306 ; Ensembl: ENSMUSG00000000001 ) is located on Mouse chromosome 3. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000000001). Exon 2~5 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit normal basal cardiac function and beta-adrenergic sensitivity. Mice homozygous for a different knock-out allele exhibit enhanced T cell migration toward CXCR3 agonists. Exon 2 starts from about 11.21% of the coding region. Exon 2~5 covers 44.44% of the coding region. The size of effective KO region: ~8075 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 9 Legends Exon of mouse Gnai3 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 5 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(30.4% 608) | C(16.65% 333) | T(29.1% 582) | G(23.85% 477) Note: The 2000 bp section upstream of Exon 2 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(28.9% 578) | C(18.95% 379) | T(30.6% 612) | G(21.55% 431) Note: The 2000 bp section downstream of Exon 5 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 4 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr3 - 108123838 108125837 2000 browser details YourSeq 116 480 939 2000 88.7% chr7 + 27237015 27313755 76741 browser details YourSeq 110 446 604 2000 89.9% chr13 - 95555319 95555662 344 browser details YourSeq 106 446 611 2000 92.2% chr3 + 33757578 33757746 169 browser details YourSeq 105 455 888 2000 87.8% chr13 + 100753546 101069343 315798 browser details YourSeq 104 449 644 2000 92.7% chr9 + 119900678 119901215 538 browser details YourSeq 103 449 612 2000 93.4% chr2 + 151792095 151792486 392 browser details YourSeq 102 446 614 2000 91.2% chr7 + 82654515 82654686 172 browser details YourSeq 99 482 609 2000 89.1% chrX - 134610714 134610842 129 browser details YourSeq 98 446 609 2000 91.0% chr2 - 101669440 101669606 167 browser details YourSeq 95 447 609 2000 92.2% chr7 + 139203106 139203274 169 browser details YourSeq 93 441 561 2000 91.9% chr10 - 40585933 40586056 124 browser details YourSeq 93 482 607 2000 91.9% chr19 + 5173696 5173821 126 browser details YourSeq 92 474 611 2000 83.4% chr4 - 3981260 3981397 138 browser details YourSeq 92 441 561 2000 89.1% chr1 + 131949517 131949635 119 browser details YourSeq 90 446 611 2000 92.5% chr16 + 30031190 30031357 168 browser details YourSeq 90 446 561 2000 92.5% chr11 + 94324384 94324503 120 browser details YourSeq 89 480 608 2000 85.3% chr2 + 37448034 37448165 132 browser details YourSeq 89 480 611 2000 84.1% chr17 + 45529071 45529203 133 browser details YourSeq 88 446 561 2000 91.6% chr11 - 79721251 79721370 120 Note: The 2000 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 2000 1 2000 2000 100.0% chr3 - 108113763 108115762 2000 browser details YourSeq 286 973 1438 2000 97.4% chr4 - 40838584 41139766 301183 browser details YourSeq 266 964 1435 2000 96.6% chr8 + 88112875 88177817 64943 browser details YourSeq 255 973 1441 2000 94.5% chr11 + 102243739 102244226 488 browser details YourSeq 250 964 1441 2000 94.6% chrX - 20460655 20461270 616 browser details YourSeq 247 976 1441 2000 95.3% chr3 - 34712155 34758708 46554 browser details YourSeq 245 974 1441 2000 88.0% chr4 - 148541182 148541521 340 browser details YourSeq 219 997 1439 2000 87.7% chr1 + 157142376 157142645 270 browser details YourSeq 209 973 1385 2000 88.7% chr16 - 16942243 16942483 241 browser details YourSeq 204 1016 1441 2000 89.3% chr18 - 36393772 36394101 330 browser details YourSeq 193 1044 1441 2000 86.6% chr10 + 81159433 81159685 253 browser details YourSeq 179 1028 1441 2000 89.9% chr16 - 44279164 44279543 380 browser details YourSeq 175 983 1441 2000 84.9% chr12 - 111661514 111661767 254 browser details YourSeq 172 974 1434 2000 87.8% chr2 + 153533090 153533390 301 browser details YourSeq 172 981 1434 2000 95.8% chr11 + 4706401 4706880 480 browser details YourSeq 169 1271 1441 2000 99.5% chr10 + 85931875 85932045 171 browser details YourSeq 168 1027 1434 2000 88.7% chr1 + 152078687 152078913 227 browser details YourSeq 166 1268 1440 2000 98.3% chr1 - 68893582 68893755 174 browser details YourSeq 166 1016 1434 2000 87.7% chr6 + 34911978 34912181 204 browser details YourSeq 164 1274 1441 2000 98.9% chr4 - 143196612 143196779 168 Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Gnai3 guanine nucleotide binding protein (G protein), alpha inhibiting 3 [ Mus musculus (house mouse) ] Gene ID: 14679, updated on 15-Oct-2019 Gene summary Official Symbol Gnai3 provided by MGI Official Full Name guanine nucleotide binding protein (G protein), alpha inhibiting 3 provided by MGI Primary source MGI:MGI:95773 See related Ensembl:ENSMUSG00000000001 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 Gnai-3; AI158965; AW537698; Galphai3 Expression Ubiquitous expression in placenta adult (RPKM 67.1), CNS E11.5 (RPKM 42.8) and 26 other tissues See more Orthologs human all Genomic context Location: 3 F2.3; 3 46.83 cM See Gnai3 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (108107275..108146152, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (107910217..107949032, complement) Chromosome 3 - NC_000069.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Gnai3 ENSMUSG00000000001 Description guanine nucleotide binding protein (G protein), alpha inhibiting 3 [Source:MGI Symbol;Acc:MGI:95773] Gene Synonyms Galphai3 Location Chromosome 3: 108,107,280-108,146,146 reverse strand. GRCm38:CM000996.2 About this gene This gene has 1 transcript (splice variant), 216 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Gnai3-201 ENSMUST00000000001.4 3262 354aa ENSMUSP00000000001.4 Protein coding CCDS17751 Q9DC51 TSL:1 GENCODE basic APPRIS P1 58.87 kb Forward strand 108.10Mb 108.11Mb 108.12Mb 108.13Mb 108.14Mb 108.15Mb Genes Gnat2-201 >protein coding (Comprehensive set... Gnat2-206 >protein coding Gnat2-204 >retained intron Gnat2-203 >protein coding Gnat2-202 >lncRNA Contigs AL671854.14 > Genes (Comprehensive set... < Gnai3-201protein coding < Gpr61-201protein coding < Gpr61-202nonsense mediated decay Regulatory Build 108.10Mb 108.11Mb 108.12Mb 108.13Mb 108.14Mb 108.15Mb Reverse strand 58.87 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 RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000000001 < Gnai3-201protein coding Reverse strand 38.87 kb ENSMUSP00000000... Low complexity (Seg) Superfamily G protein alpha subunit, helical insertion P-loop containing nucleoside triphosphate hydrolase SMART Guanine nucleotide binding protein (G-protein), alpha subunit Prints G-protein alpha subunit, group I Guanine nucleotide binding protein (G-protein), alpha subunit Pfam Guanine nucleotide binding protein (G-protein), alpha subunit PANTHER PTHR10218:SF230 Guanine nucleotide binding protein (G-protein), alpha subunit Gene3D 3.40.50.300 G protein alpha subunit, helical insertion CDD Guanine nucleotide binding protein (G-protein), alpha subunit All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 354 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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