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Mouse Fam19a5 Knockout Project (CRISPR/Cas9)

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Mouse Fam19a5 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Fam19a5 Knockout Project (CRISPR/Cas9) Objective: To create a Fam19a5 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fam19a5 gene (NCBI Reference Sequence: NM_001252310 ; Ensembl: ENSMUSG00000054863 ) is located on Mouse chromosome 15. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000230414). Exon 2 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: Exon 2 starts from about 28.54% of the coding region. Exon 2 covers 37.88% of the coding region. The size of effective KO region: ~150 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 gRNA region 5' gRNA region 3' 1 2 4 Legends Exon of mouse Fam19a5 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 150 bp section 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 150 bp section 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. Page 3 of 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(150bp) | A(22.0% 33) | C(30.0% 45) | T(13.33% 20) | G(34.67% 52) Note: The 150 bp section 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(150bp) | A(22.0% 33) | C(30.0% 45) | T(13.33% 20) | G(34.67% 52) Note: The 150 bp section 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. 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 150 1 150 150 100.0% chr15 + 87681482 87681631 150 Note: The 150 bp section 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 150 1 150 150 100.0% chr15 + 87681480 87681629 150 browser details YourSeq 21 17 40 150 95.9% chr11 + 53708591 53708615 25 Note: The 150 bp section of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Tafa5 TAFA chemokine like family member 5 [ Mus musculus (house mouse) ] Gene ID: 106014, updated on 24-Oct-2019 Gene summary Official Symbol Tafa5 provided by MGI Official Full Name TAFA chemokine like family member 5 provided by MGI Primary source MGI:MGI:2146182 See related Ensembl:ENSMUSG00000054863 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 Tafa-5; Tara-5; Fam19a5 Summary This gene is a member of the TAFA family which is composed of five highly homologous genes that encode small secreted Expression proteins. These proteins contain conserved cysteine residues at fixed positions, and are distantly related to MIP-1alpha, a member of the CC-chemokine family. The TAFA proteins are predominantly expressed in specific regions of the brain, and are postulated to function as brain-specific chemokines or neurokines, that act as regulators of immune and nervous cells. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Nov 2011] Orthologs Biased expression in frontal lobe adult (RPKM 18.4), cerebellum adult (RPKM 17.8) and 14 other tissues See more human all Genomic context Location: 15; 15 E3 See Tafa5 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (87544219..87759364) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (87455660..87589794) Chromosome 15 - NC_000081.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Tafa5 ENSMUSG00000054863 Description TAFA chemokine like family member 5 [Source:MGI Symbol;Acc:MGI:2146182] Gene Synonyms Fam19a5, Tara-5 Location Chromosome 15: 87,544,299-87,759,364 forward strand. GRCm38:CM001008.2 About this gene This gene has 2 transcripts (splice variants), 225 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tafa5-201 ENSMUST00000068088.7 2603 125aa ENSMUSP00000064808.6 Protein coding CCDS49693 Q91WE9 TSL:1 GENCODE basic Tafa5-202 ENSMUST00000230414.1 2649 132aa ENSMUSP00000155322.1 Protein coding - Q91WE9 GENCODE basic APPRIS P1 235.07 kb Forward strand 87.55Mb 87.60Mb 87.65Mb 87.70Mb 87.75Mb Genes (Comprehensive set... Tafa5-202 >protein coding Tafa5-201 >protein coding Contigs AC124381.4 > < AC124449.3 AC123925.4 > Regulatory Build 87.55Mb 87.60Mb 87.65Mb 87.70Mb 87.75Mb Reverse strand 235.07 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000230414 215.06 kb Forward strand Tafa5-202 >protein coding ENSMUSP00000155... Transmembrane heli... Pfam Chemokine-like protein TAFA PANTHER PTHR31878:SF0 Chemokine-like protein TAFA-5 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 132 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
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