B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm2(tetO-GFP/RNAi:Rpl5)Begg}/J

Stock number: 034150
Product name: shRNA Rpl5

Research areas: Hematological Research, Research Tools

Description

These shRNA Rpl5/rtTA GFP reporter mice provide doxycycline-mediated, reversible expression of Rpl5 (ribosomal protein L5) short hairpin RNA and can be used to downregulate Rpl5 expression by RNA interference.  These mice may be useful in studying Diamond-Blackfan anemia (DBD).

Detailed description

The Rpl5 (ribosomal protein L5) gene encodes a protein that binds 5S rRNA to form a stable complex called the 5S ribonucleoprotein particle (RNP). The complex is required for the transport of nonribosome-associated cytoplasmic 5S rRNA to the nucleolus for assembly into ribosomes. Mutations in Rpl5 are associated with Diamond-Blackfan anemia (DBD), a congenital bone marrow disorder inherited in autosomal dominant fashion. The shRNA Rpl5 allele is a Col1a1 GFP knock-in allele designed to allow Tet-controllable/reversible expression of an shRNA that targets/silences Rpl5 expression as well as GFP reporter expression. When combined with a reverse transactivator under the control of the widespread CAG promoter (R26-M2rtTA) and administration of doxycycline, activation of Rpl5 shRNA reduces Rpl5 expression by three fold in CD71^high Ter119^high bone marrow cells. Mice homozygous for Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} and heterozygous for Col1a1^{tm1(tetO-GFP/RNAi:Rpl5)Beggs} are viable and fertile. Following 2 weeks of treatment with doxycycline, shRNA Rpl5/rtTA mice develop mild anemia (20%), reticulocytopenia and bone marrow erythroblastopenia. Mice homozygous for shRNA Rpl5 did not survive doxycycline treatment. These mice may be useful in studying Diamond-Blackfan anemia (DBD).

Of note, a non-specific shRNA control line is available as B6.129-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm1(tetO-GFP/RNAi:rluc)Beggs}/J (Stock No. 034380).

Development

RMCE (recombinase-mediated cassette exchange) was utilized to insert tetO-GFP/RNAi:Rpl5 into the Col1a1 locus. KH2 embryonic stem cells (C57BL/6 x 129S4Sv/Jae), carrying the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele (see Stock No. 006965), were previously re-targeted to insert an FRT-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette in the 3' UTR of the Col1a1 locus, thus adapting the ES cell line for RMCE. The vector contains a tetracycline responsive promoter (TRE;tetO) controlling expression of a turboGFP sequence (tGFP; a fast-maturating and short-half-life green fluorescent protein variant) and short-hairpin RNA sequence targeting Rpl5 embedded in the MIR30 structure. The vector was then co-electroporated with a FLPe recombinase-expressing vector into the RMCE-ready KH2 ES cells. The resulting correctly targeted ES cells, carrying rtTA*M2 in the Gt(ROSA)26Sor locus and tetO-GFP/RNAi:Rpl5 in the Col1a1 locus, were injected into blastocysts. The resulting chimeric animals were bred to C57BL/6 mice. The colony then was backcrossed to C57BL/6 for 2-3 generations. Upon arrival, mice were bred to C57BL/6J for at least 1 generation to establish the colony.

Allele

Symbol: Col1a1^{tm2(tetO-GFP/RNAi:Rpl5)Begg}
Name: targeted mutation 2, Alan Beggs
MGI accession ID: MGI:6368192
Generation method: Targeted
Attributes: Reporter; Inducible; Knockdown
Synonyms: shRNA Rpl5
Marker symbol: Col1a1
Marker name: collagen, type I, alpha 1
Marker synonyms: CAFYD; Col1a-1; Cola1; Cola-1; COLIA1; EDSARTH1; EDSC; Mov-13; OI1; OI2; OI3; OI4
Chromosome: 11
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Site of expression: TetO-inducible
Molecular note: RMCE (recombinase-mediated cassette exchange) was utilized to insert tetO-GFP/RNAi:Rpl5 into the Col1a1 locus. The vector contains a tetracycline responsive promoter (TRE;tetO) controlling expression of a turboGFP sequence (tGFP; a fast-maturating and short-half-life green fluorescent protein variant) and short-hairpin RNA sequence targeting Rpl5 embedded in the MIR30 structure. The vector was then co-electroporated with a FLPe recombinase-expressing vector into the RMCE-ready KH2 ES cells.
General note: The allele was made in KH2 cells that carry Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} and Col1a1^{tm13(neo/hygro*)Jae}. This allele was generated with Col1a1^{tm13(neo/hygro*)Jae}.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}
Name: targeted mutation 1, Rudolf Jaenisch
MGI accession ID: MGI:3702294
Generation method: Targeted
Attributes: Transactivator
Synonyms: Gt(ROSA)26Sor^{tm1(M2rtTA)Jae}; M2; M2-rtTA; R26-M2rtTA; R26-rtTA; Rosa26-M2rtTA
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli
Site of expression: Expresses an optimized rtTA protein (rtTA-M2). Inducible target gene expression is detected in liver, bone marrow, stomach, intestine, and skin, with lower levels in the heart, lungs, kidney, spleen, and thymus; no expression is detected in the brain and testes.
Molecular note: An optimized form of reverse tetracycline controlled transactivator (rtTA-M2) was inserted downstream of the Gt(ROSA)26Sor promoter and was followed by a PGK-puro selection cassette. This mutant form of rtTA termed M2 has five amino acid substitutions in the tetR moiety of tTA: S12G, E19G, A56P, D148E and H179R. This mutated form of transactivator protein has increased doxycycline sensitivity. Mice have widespread expression of the rtTA-M2 protein.
General note: This mutation was created during the generation of mutant ES cell line KH2, which also contains a frt docking site downstream of the endogenous Col1a1 locus on Chr 11 (Col1a1). The two mutations have subsequently been bred apart.