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Mouse Gjb5 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Gjb5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Gjb5 gene (NCBI Reference Sequence: NM_010291 ; Ensembl: ENSMUSG00000042357 ) is located on Mouse chromosome 4. 2 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000046498). 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 Gjb5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-69I2 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 null allele exhibit some embryonic lethality, reduced weight and reduced placental development.
Exon 2 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 2. The size of effective cKO region: ~2315 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele 5' gRNA region gRNA region 3'
1 2 1 2 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Homology arm Exon of mouse Gjb5 cKO region Exon of mouse Gjb4 loxP site
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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. 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 GC Content Distribution Window size: 300 bp
Sequence 12
Summary: Full Length(6813bp) | A(26.76% 1823) | C(26.61% 1813) | T(21.25% 1448) | G(25.38% 1729)
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.
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BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr4 - 127356350 127359349 3000 browser details YourSeq 61 302 687 3000 74.7% chr1 - 185218852 185219176 325 browser details YourSeq 54 290 472 3000 88.5% chr11 + 119092897 119093083 187 browser details YourSeq 46 237 357 3000 84.4% chr12 - 76879876 76879992 117 browser details YourSeq 39 246 352 3000 93.4% chr8 + 124226216 124226323 108 browser details YourSeq 35 443 479 3000 97.3% chr9 - 12436163 12436199 37 browser details YourSeq 35 252 352 3000 61.6% chr19 - 11990156 11990229 74 browser details YourSeq 35 233 353 3000 70.8% chr17 + 52284099 52284200 102 browser details YourSeq 34 252 315 3000 87.0% chr18 - 66394029 66394097 69 browser details YourSeq 34 252 314 3000 75.0% chr17 - 32448076 32448129 54 browser details YourSeq 34 284 357 3000 97.3% chr11 - 114328528 114328602 75 browser details YourSeq 34 839 877 3000 97.3% chr1 - 153887118 153887190 73 browser details YourSeq 32 826 873 3000 79.0% chr12 + 24443801 24443844 44 browser details YourSeq 31 301 357 3000 94.3% chr19 - 41819529 41819586 58 browser details YourSeq 29 286 318 3000 94.0% chr16 - 93600629 93600661 33 browser details YourSeq 29 445 473 3000 100.0% chr7 + 58319330 58319358 29 browser details YourSeq 28 281 314 3000 91.2% chr3 - 32699689 32699722 34 browser details YourSeq 28 444 471 3000 100.0% chr9 + 21024649 21024676 28 browser details YourSeq 28 827 863 3000 77.5% chr10 + 99394286 99394317 32 browser details YourSeq 27 836 868 3000 90.0% chr13 - 112074955 112074986 32
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% chr4 - 127352537 127355536 3000 browser details YourSeq 258 2154 2596 3000 91.8% chr10 - 24626101 24962609 336509 browser details YourSeq 225 2170 2595 3000 91.0% chr1 + 134391990 134392453 464 browser details YourSeq 217 2152 2596 3000 93.0% chr16 + 76564264 76564820 557 browser details YourSeq 204 2185 2591 3000 85.8% chr3 - 69412742 69413086 345 browser details YourSeq 202 2152 2593 3000 89.0% chr1 + 135237785 135238217 433 browser details YourSeq 192 2156 2475 3000 95.4% chr1 - 22941469 23176322 234854 browser details YourSeq 190 2150 2587 3000 86.1% chr1 - 189570617 189570905 289 browser details YourSeq 187 2150 2596 3000 83.9% chr1 + 183525016 183525339 324 browser details YourSeq 185 2149 2596 3000 86.8% chr8 - 24363614 24363967 354 browser details YourSeq 179 2176 2593 3000 85.2% chr8 - 90706219 90706570 352 browser details YourSeq 178 2150 2593 3000 90.0% chr13 + 52412929 52413362 434 browser details YourSeq 176 2174 2600 3000 85.4% chr1 + 23746689 23747005 317 browser details YourSeq 168 2189 2587 3000 90.8% chr18 + 55041750 55042171 422 browser details YourSeq 165 2152 2596 3000 85.7% chr10 - 24626208 24626434 227 browser details YourSeq 164 2152 2593 3000 85.0% chr14 + 73472061 73472325 265 browser details YourSeq 162 2152 2475 3000 87.6% chr10 - 24626152 24626460 309 browser details YourSeq 161 2149 2593 3000 86.7% chr1 - 187815485 187815842 358 browser details YourSeq 156 2191 2541 3000 88.9% chr5 - 118910664 118911623 960 browser details YourSeq 155 2152 2547 3000 82.1% chr1 + 132743762 132744021 260
Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Gjb5 gap junction protein, beta 5 [ Mus musculus (house mouse) ] Gene ID: 14622, updated on 12-Aug-2019
Gene summary
Official Symbol Gjb5 provided by MGI Official Full Name gap junction protein, beta 5 provided by MGI Primary source MGI:MGI:95723 See related Ensembl:ENSMUSG00000042357 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Gjb-5; Cx31.1; Cnx31.1 Expression Biased expression in placenta adult (RPKM 18.0), colon adult (RPKM 3.2) and 4 other tissues See more Orthologs human all
Genomic context
Location: 4 D2.2; 4 61.51 cM See Gjb5 in Genome Data Viewer
Exon count: 2
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (127354809..127358164, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (127032053..127035408, complement)
Chromosome 4 - NC_000070.6
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Transcript information: This gene has 1 transcript
Gene: Gjb5 ENSMUSG00000042357
Description gap junction protein, beta 5 [Source:MGI Symbol;Acc:MGI:95723] Gene Synonyms Cnx31.1, Cx31.1, Gjb-5, connexin 31.1 Location Chromosome 4: 127,354,809-127,358,181 reverse strand. GRCm38:CM000997.2 About this gene This gene has 1 transcript (splice variant), 343 orthologues, 19 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Gjb5-201 ENSMUST00000046498.2 1721 271aa ENSMUSP00000045325.2 Protein coding CCDS18670 Q02739 Q542M8 TSL:1 GENCODE basic APPRIS P1
23.37 kb Forward strand 127.345Mb 127.350Mb 127.355Mb 127.360Mb 127.365Mb Contigs AL626768.21 > Genes (Comprehensive set... < Gjb4-201protein coding < Gjb5-201protein coding
< Gjb4-202protein coding
Regulatory Build
127.345Mb 127.350Mb 127.355Mb 127.360Mb 127.365Mb Reverse strand 23.37 kb
Regulation Legend CTCF Promoter Promoter Flank
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
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Transcript: ENSMUST00000046498
< Gjb5-201protein coding
Reverse strand 3.37 kb
ENSMUSP00000045... Transmembrane heli... Low complexity (Seg) SMART Connexin, N-terminal Gap junction protein, cysteine-rich domain
Prints Gap junction beta-5 protein (Cx30.3)
Connexin Pfam Connexin, N-terminal PROSITE patterns Connexin, conserved site Connexin, conserved site
PANTHER Connexin
PTHR11984:SF29 Gene3D Connexin, N-terminal domain superfamily
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant synonymous variant
Scale bar 0 40 80 120 160 200 271
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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