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Mouse Rab11fip2 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Rab11fip2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Rab11fip2 gene (NCBI Reference Sequence: NM_001164367 ; Ensembl: ENSMUSG00000040022 ) is located on Mouse chromosome 19. 5 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 5 (Transcript: ENSMUST00000171986). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rab11fip2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-151E24 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:
Exon 2~3 is not frameshift exon, and covers 75.59% of the coding region. The size of intron 1 for 5'-loxP site insertion: 6079 bp, and the size of intron 3 for 3'-loxP site insertion: 22157 bp. The size of effective cKO region: ~2325 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 3 5 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Rab11fip2 Homology arm cKO region 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(8752bp) | A(28.76% 2517) | C(18.69% 1636) | T(32.43% 2838) | G(20.12% 1761)
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% chr19 - 59937608 59940607 3000 browser details YourSeq 63 136 517 3000 71.9% chr10 - 126753694 126753870 177 browser details YourSeq 46 1127 1207 3000 91.4% chr11 - 56256006 56256089 84 browser details YourSeq 34 1129 1170 3000 94.8% chr18 + 11463270 11463313 44 browser details YourSeq 33 473 511 3000 92.4% chr6 - 96011121 96011159 39 browser details YourSeq 33 473 510 3000 94.6% chr17 + 43792288 43792422 135 browser details YourSeq 31 473 509 3000 91.9% chr6 - 145521519 145521555 37 browser details YourSeq 30 479 512 3000 94.2% chr9 - 74504029 74504062 34 browser details YourSeq 30 481 519 3000 85.3% chr15 + 64561595 64561631 37 browser details YourSeq 25 1164 1193 3000 93.2% chr1 + 128749216 128749247 32 browser details YourSeq 23 1208 1231 3000 100.0% chr1 - 59304776 59304801 26 browser details YourSeq 23 488 511 3000 100.0% chr16 + 15240316 15240341 26 browser details YourSeq 21 489 511 3000 95.7% chr12 + 35236831 35236853 23
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% chr19 - 59932356 59935355 3000 browser details YourSeq 24 1124 1152 3000 96.2% chr2 + 102068387 102068416 30 browser details YourSeq 20 2462 2487 3000 88.5% chr12 - 89346021 89346046 26
Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Rab11fip2 RAB11 family interacting protein 2 (class I) [ Mus musculus (house mouse) ] Gene ID: 74998, updated on 12-Aug-2019
Gene summary
Official Symbol Rab11fip2 provided by MGI Official Full Name RAB11 family interacting protein 2 (class I) provided by MGI Primary source MGI:MGI:1922248 See related Ensembl:ENSMUSG00000040022 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 Nrip11; AW558126; 4930470G04Rik; A830046J09Rik Expression Broad expression in cortex adult (RPKM 5.3), CNS E18 (RPKM 5.1) and 25 other tissues See more Orthologs human all
Genomic context
Location: 19; 19 D3 See Rab11fip2 in Genome Data Viewer
Exon count: 8
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (59893502..59943656, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (59978787..60019557, complement)
Chromosome 19 - NC_000085.6
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Transcript information: This gene has 3 transcripts
Gene: Rab11fip2 ENSMUSG00000040022
Description RAB11 family interacting protein 2 (class I) [Source:MGI Symbol;Acc:MGI:1922248] Gene Synonyms 4930470G04Rik, A830046J09Rik, Rab11-FIP2, nRip11 Location Chromosome 19: 59,902,884-59,943,654 reverse strand. GRCm38:CM001012.2 About this gene This gene has 3 transcripts (splice variants), 197 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Rab11fip2-203 ENSMUST00000171986.7 5363 370aa ENSMUSP00000128813.1 Protein coding CCDS50482 G3UW57 TSL:1 GENCODE basic
Rab11fip2-201 ENSMUST00000051996.12 4482 529aa ENSMUSP00000059978.6 Protein coding CCDS38033 G3XA57 TSL:1 GENCODE basic
Rab11fip2-202 ENSMUST00000170819.1 1539 512aa ENSMUSP00000133151.1 Protein coding - G3XA57 TSL:5 GENCODE basic APPRIS P1
60.77 kb Forward strand 59.90Mb 59.91Mb 59.92Mb 59.93Mb 59.94Mb 59.95Mb Genes Gm4219-201 >lncRNA (Comprehensive set...