Generation and characterization of a novel transgenic mouse harboring conditional nuclear factor-kappa B/RelA knockout alleles
© The Author(s). 2016
Received: 19 May 2016
Accepted: 13 September 2016
Published: 23 September 2016
Nuclear Factor-Kappa B (NF-kB) is a family of transcription factors that are important in embryonic development, inflammation, epithelial-to-mesenchymal transition and cancer. The 65 kDa RelA subunit is the major transcriptional activator of the NF-kB pathways. Whole-body deficiency of RelA leads to massive apoptosis of liver hepatocytes and death in utero. To study the role of RelA in physiology and in disease states in a manner that circumvents this embryonic lethal phenotype, we have generated a mouse with RelA conditional knockout (CKO) alleles containing loxP sites that are deleted by activated Cre recombinase.
We demonstrate that RelACKO/CKO mice are fertile, do not display any developmental defects and can be crossed with Cre-expressing mice to delete RelA in a temporal, tissue-specific manner. Our mating of RelACKO/CKO mice with Zp3-Cre transgenic led to embryonic lethality of RelA-deficient embryos. In contrast, mating of RelACKO/CKO mice with Col1α2-CreER mice allowed for the generation of double transgenics which could be stimulated with tamoxifen to induce fibroblast-specific RelA deletion in adulthood.
Based on our collective data, we conclude that this novel RelACKO/CKO mouse allows for efficient deletion of RelA in a tissue-specific manner. This RelACKO/CKO mouse will be an invaluable tool for deciphering the mechanistic roles of RelA in various cells and tissues during development and in disease.
KeywordsNF-kB RelA p65 Flox Tamoxifen Cre Col1a2
NF-kB is an inducible transcription factor complex involved in the regulation of genes necessary for cell survival, differentiation, immunity and inflammation . RelA, C-Rel and RelB are transcriptional activating subunits that contain a N-terminal Rel homology domain (RHD) and a C-terminal transcription activation domain (TAD) whereas NF-kB1 (p50/p105) and NF-kB2 (p52/p100) are DNA-binding proteins that contain C-terminal autoinhibitory ankyrin repeat domains . In most cells, NF-kB is high molecular weight heterodimeric complexes containing a DNA-binding- and a transcription activator subunit retained in the cytoplasm by Inhibitor of kappa B (IkB) proteins. Receptor activation by numerous ligands induces phosphorylation of IkBα, targeting it for degradation and allowing nuclear translocation of RelA•NF-kB1 dimers, the most abundant and most potent transcription factor pair required for activation of inflammatory and anti-apoptotic gene expression programs .
Beg and colleagues were the first to describe the lethality of whole-body RelA deficiency in mice . Mating of RelA heterozygous mice failed to generate any RelA-deficient (RelA-/-) animals and an analysis of embryos indicated that RelA-/- embryos undergo massive liver degeneration with hepatocyte apoptosis and consequently die in utero. These findings were independently corroborated using a different gene-targeting vector . This study demonstrated that when RelA-/- embryonic livers are transplanted into SCID mice, T and B cells populate spleen and lymph nodes to similar extent as wild-type (WT) cells, suggesting that the lethal effect of RelA-/- in utero is due to impairment of hepatocyte development and not due to deranged hematopoiesis. In a subsequent finding, the lethal phenotype of RelA-/- was rescued by absence of TNFα . RelA activation downstream of the TNF-receptor is necessary for the production of anti-apoptotic molecules, including TRAF-1, TRAF-2, c-IAP1 AND c-IAP2 that protect cells from local, endogenously produced TNFα-induced apoptosis [7, 8].
To overcome embryonic lethality produced by whole-body RelA deletion and to investigate the role of RelA in disease-specific states, investigators have generated transgenic mice inserting loxP sites in the RelA gene (RelA-floxed). DNA recombination mediated by Cre recombinase in RelA-floxed animals leads to truncation or deletion of the RelA protein. Algul and colleagues created a RelA-floxed mouse containing loxP sites in RelA introns 6 and 10 . Induction of Cre led to deletion of exons 7–10, encoding the RHD, producing a truncated RelA that failed to translocate to the nucleus. This strategy results in expression of a truncated RelA C-terminal TAD that potentially confounds the study. Recently, another group created RelA-floxed mice with loxP sites flanking the promoter region and exon 1 that resulted in the deletion of RelA in B-cells when RelA-floxed animals were crossed with CD19-Cre trasngenics . The strategy to excise only exon 1 of the RelA gene has the potential to allow the expression of alternatively spliced variants. In the case of RelA, there is putative internal ribosome entry site (IRES) in exons 4–5 which allows for 5′cap-indepedant translation . Additionally, there is evidence of alternatively spliced variants of RelA in different cell types. For example, p65Δ variant lacks aa 222–231 and is abundantly expressed in pre-B and erythroid colony forming cells . During screening of human adult osteoblastic cDNA library a RelA variant, p65Δ2, has been found that lacks amino acids (aa) 13–25 and 506 . The N-terminus aa 13–25 are part of the RHD, corresponding to exon 2 and 3, and may be needed for binding to DNA. Although there has not been any exhaustive investigation into alternative RelA splicing variants, the presence of IRES in exons 4–5 and existence of RelA NH2-terminal splice variants suggests that some RelA transcripts may be produced in specialized cells under pathophysiological conditions that lack exon 1 but still can undergo translation. These splice variants would not be targeted in the flox mouse created by Heise and colleagues. Furthermore, cells that undergo recombination in the transgenic by Hesie et al. begin expressing eGFP. This strategy makes it easier to track recombined cells but the eGFP signal could be confounding in leukocytes, such as monocytes/macrophages, that have significant intracellular granules and are autofluorescent in the same emission range as GFP . We decided to create a RelA-floxed mouse for investigating NF-kB signaling in vivo because, at the time, no such experimental tool was commercially available. Our group has developed a RelA-floxed transgenic mouse that allows for complete deletion of RelA and which will be made available for use by the scientific community.
Results and Discussion
In summary, we report the generation of a transgenic mouse containing RelA-flox alleles that recombine in the presence of active Cre recombinase to generate RelA-/- cells. This will be an important tool for studies investigating RelA signaling in adult tissues since it allows one to bypass a major limitation, death in utero, that accompanies whole-body RelA-/-. Furthermore, this will be a valuable mouse model for exploring the role of RelA in development, an under-explored area due to a lack of appropriate tools for investigation.
Construction of the RelA conditional knockout vector
A 7.2-kb AfeI-HindIII DNA fragment (RelA 5′ upstream–intron 8 region) was isolated from a bacterial artificial chromosome (BAC) clone, RP24-329M16 (obtained from the Children’s Hospital Oakland Research Institute), and subcloned into pBluescript II SK (-) (Agilent, Santa Clara, CA) between SmaI and HindIII sites. A PGKneobpA cassette  flanked by an frt site on one end and an frt-loxP sequence on the other was inserted into the unique EcoRV site in intron 4 in reverse orientation relative to the orientation of RelA transcription via blunt-end ligation. Oligonucleotides for creating a DNA fragment containing a loxP site with an inserted Asp718 site and two AflII ends were synthesized and annealed, and inserted into a unique AflII site in intron 8. Finally, MC1tkpA cassette was added into the SalI site of the multiple cloning site region of the pBluescript for the enrichment of homologous recombinants via negative selection .
Genetic engineering of mouse embryonic stem (ES) cells
The targeting vector was propagated, purified by cesium banding, and linearized by NotI. A total of 1 × 107 cells suspended in DPBS was electroporated with 25 μg DNA using Gene Pulser (500 μF/230 V, BIO-RAD, Hercules, CA). The cells were then cultured in 200 μg/ml Geneticin (G418) and 200 nM 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU) for 9 days. ES cell colonies that survived the G418-FIAU drug selection were expanded in three sets of 96-well plates. One set of the cells was cryopreserved for blastocyst injection; the other two sets were processed for Southern blot analysis. The 5′ and 3′ flanking probes were hybridized to EcoRV-digested DNA and Asp718-digested DNA, respectively.
Chimera production and animals
Correctly targeted ES cell clones were injected into C57BL/6J blastocysts and injected embryos were transferred to 2.5-day-post-coitum pseudo-pregnant Swiss Webster female mice. All animal work was approved by UTMB’s IACUC committee.
Genotyping of the mouse using genomic Southern blot
Mice were genotyped by Southern blot analysis of tail DNA with 5′-flanking, 3′-flanking, and/or internal probes. All the probe fragments were generated by PCR, and subcloned into a T-vector. 5′-flanking probe: A 529-bp DNA was amplified form the BAC DNA with forward primers, TTGTGGGTAGCTGTGGTCAA, and reverse primer, CCAGCACTCCAGAAGAAAGG. 3′-flanking probe: A 468-bp DNA was amplified from the BAC DNA with forward primers, GGGAGAAGTGCAGCCCGGC, and reverse primer, CCCGGCCTCCCCCTGAGAA. Internal probe 1: A 561-bp DNA was amplified from the BAC DNA with forward primers, GATCCAGTGTGTGAAGAAGC, and reverse primer, GGTTATCAAAAATCGGATGT. Internal probe 2: A 209-bp DNA was amplified from a pool of cDNA originating from mouse embryonic fibroblast RNA with forward primers, GATCCAGTGTGTGAAGAAGC, and reverse primer, GGTTATCAAAAATCGGATGT. Genomic DNA digested by appropriate restriction enzymes was separated on agarose/TAE gels, and blotted onto Hybond-XL membranes (GE Healthcare, Waukesha, WI). Blots were hybridized at 65 °C overnight with radiolabeled and pre-associated probes in 1 M sodium chloride, 1 % SDS, 10 % dextran sulfate with 100 μg/ml salmon sperm DNA; and washed at 65 °C in 0.2X SSC, 0.1 % SDS.
Generation of MEF cells and genotyping of the mouse using PCR
MEF cells were generated as described previously . DNA was isolated from tail biopsies or MEF cells using phenol:chloroform extraction and ethanol precipitation followed by PCR. Briefly, tail biopsies or 500K cells were incubated with 700 μl of digestion buffer (50 mM Tris-HCl, pH 8; 100 mM EDTA; 100 mM NaCl; 1 % SDS; 350 μg Proteinase K) overnight at 55 °C. Next morning, 700 μl phenol:cholorform (Sigma) was added to each sample, samples were vortexed and centrifuged at 13,000 RPM for 10 min. Approximately 500 μl of supernatant was collected and 2× as much ethanol was added to precipitate the DNA. The mixture was centrifuged, the supernatant discarded and DNA pellet was re-suspended in 50 μl of TE buffer. PCR reaction was set up using the following primers: 5–8Δ F, GCCGGCCAGGCAGCTCTTAC, and 5–8 Δ R, GGCCAGTCACCATGGCCAGC, provide a 335 bp product only when RelACKO allele undergoes recombination and 7–8 F, ACACTGCCGAGCTCAAGATC, and 7–8 R, AGCTGCATGGAGACTCGAAC, provide a 425 bp product when WT RelA is present. Presence of RelACKO alleles was determined by RelAFlx F, TGCAAACAGACCTCCTTTGTCTTGA, and RelAFlx R, TCCTGAGACCAGACTCCTCCTCC, primers which provides a 450 bp product if the CKO allele is present or 270 bp product for a WT allele. PCR reaction was denatured at 94 °C for 2 min and subjected to 35 cycles of 30 s at 94 °C, 30 s at 54 °C, and 60 s at 72 °C. The PCR products were separated on a 1 % agarose gel containing ethidium bromide and imaged on a UV transilliumintor. Col1a2-CreER mice were genotyped according to instructions by The Jackson Laboratory (stock no. 016237).
Quantitative RT-PCR (QRT-PCR)
RNA was extracted from MEF cells using Trizol Reagent (Life Technologies, 15596-026) and was quantified using NanoDrop 2000 (Thermo Scientific). 1 μg RNA was reverse transcribed to cDNA using SuperScript III First-Strand Synthesis System in a 20 μl reaction according to the manufacturer’s instruction (Invitrogen, 18080-51). cDNA was diluted 1:2 and 3 μl of the product was used in a 30 μl reaction mixture containing 15 μl SybrGreen mix and 500 nM final concentration of RelA forward, CCGGGATGGCTACTATGAGG, and RelA reverse, TCTTCACACACTGGATCCCC, primers or 18s rRNA forward, AGTCCCTGCCCTTTGTACACA, and 18s rRNA reverse, CGATCCGAGGGCCTCACTA, primers. The reaction mixtures were aliquoted into a Bio-Rad 96-well PCR plate and sealed. The plates were denatured at 95 °C for 3 min followed by 40 cycles of 15 s at 95 °C, 60 s at 60 °C and 1 min at 72 °C. PCR products were subjected to melting curve analysis to ensure that a single product was produced. Change in gene expression was determined using ΔΔCT method. RelA mRNA was normalized to 18s rRNA.
Mouse embryos were formalin fixed and paraffin embedded. Tissue sections (6 μm) were deparaffinized and rehydrated; antigen retrieval was performed with 10 mM Sodium Citrate, pH6 before blocking with 5 % goat serum and incubation with anti-RelA C-terminus (Santa Cruz sc-3702, 1:200, or Abcam ab7970, 1:300), anti-RelA N-terminus (Santa Cruz sc-109, 1:100), anti-cleaved capase 3 (Cell Signaling 5A1E, 1:100) or anti-cleaved PARP antibody (Cell Signaling D64E10, 1:50) overnight at 4 °C. A biotinylated secondary antibody and avidin-biotin complex (Vector Labs, PK6101) was used to amplify the signal and DAB substrate (Vector Labs, SK4100) was used to detect the antigen-antibody complex. Images were obtained at 600× magnification using a Nikon digital camera DXM1200F attached to a Nikon Eclipse 80i microscope.
Semiquantitative Western blot
Whole cell protein lysates were extracted from MEF cells using RIPA buffer containing protease inhibitor cocktail. The resultant protein mixture was fractionated by 10 % SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. After blocking with 5 % milk in TBS-tween buffer, the membrane was incubated with rabbit anti-RelA antibody (Santa Cruz sc-372, 1:1000) and mouse anti-beta actin (Sigma A5316, 1:10,000) overnight at 4 °C. Membranes were washed with TBS-tween before being incubated with IRDye 800-conjugated anti-rabbit and IRDye 700 conjugated anti-mouse secondary antibodies. RelA and beta-actin bands were detected using Licor Odyssey infrared scanner. Band densities were quantified using Image J (NIH) and represented in arbitrary units.
Tamoxifen mediated genetic recombination and fibroblast culture
RelACKO/CKO female mice were crossed with Col1α2-CreER male mice (gift from Dr. Arjun Deb, UNC Chapel Hill), both on C57Bl/6 background, for three generations to generate RelACKO/CKO Cre + and RelACKO/CKO Cre- animals. Adult male Cre + and Cre- mice, 3–4 weeks old, were injected with Tamoxifen (Sigma T5648) 1 mg/day intraperitoneally for 10 days. Tamoxifen was dissolved in 10 % ethanol and 90 % corn oil for 10 mg/ml working solution. After another 3 weeks, mice were euthanized and tissues were harvested for characterization. Skin samples were incubated with 0.25 % trypsin overnight at 4 °C. The skin was then minced and digested in 0.14 Wunsch units/ml Liberase Blendzyme 3 (Roche) containing 1× antibiotic/antimycotic (Invitrogen) in DMEM/F12 media for 1–2 h at 37 °C. The skin was further dissociated and resuspended in DMEM/12 media containing 10 % FBS, 1× antibiotic/antimycotic and nonessential amino acids (complete media). Cells were centrifuged at 1000×g for 10 mins and the pellet was resuspended in complete media for culturing. After 3 passages, only surviving cells were dermal fibroblasts which were characterized via immunofluorescence, QRT-PCR and Western blot.
Student’s t-test (2-tail, assuming unequal variance) was use to analyze difference between two groups. P < 0.05 was considered statistically significant.
Nuclear factor-kappa B
Rel homology domain
Transcription activation domain
We would like to thank Dr. Tom Wood, Dr. Iryna Pinchuk and Dr. Jun Yang for helpful discussions. Core laboratory support was provided by the Transgenic Mouse Facility and Recombinant DNA Laboratories of UTMB.
This work was supported by F30HL128036 (T.I.), PO1 AI068865 (A.R.B.), UTMB CTSA UL1TR001439 (A.R.B.), NIEHS P30 ES006676 (A.R.B.), and AHA 13GRNT17120070 (R.G.T.).
Availability of data and materials
The data on which this manuscript draws its conclusion is provided in the body of the manuscript.
TI, MW, and ARIII helped create the targeting vector and the conditional knockout allele mouse. TI and HS maintained the animal colonies and helped characterized the global knock out and fibroblast-specific knock out mouse. ARB and RGT conceived of the study, contributed to the experimental design, data interpretation and coordinated the project. All listed authors were involved in preparing the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The University of Texas Medical Branch’s Institutional Animal Care and Use Committee approved the animal work that is reported in this manuscript (protocol# 0105020B).
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- Baker RG, Hayden MS, Ghosh S. NF-kB, inflammation, and metabolic disease. Cell Metab. 2011;13:11–22.View ArticlePubMedPubMed CentralGoogle Scholar
- Gilmore TD. Introduction to NF-kappaB: players, pathways, perspectives. Oncogene. 2006;25:6680–4.View ArticlePubMedGoogle Scholar
- Brasier AR. The nuclear factor-kappaB-interleukin-6 signalling pathway mediating vascular inflammation. Cardiovasc Res. 2010;86:211–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature. 1995;376:167–70.View ArticlePubMedGoogle Scholar
- Doi TS, Takahashi T, Taguchi O, Azuma T, Obata Y. NF-kappa B RelA-deficient lymphocytes: normal development of T cells and B cells, impaired production of IgA and IgG1 and reduced proliferative responses. J Exp Med. 1997;185:953–61.View ArticlePubMedPubMed CentralGoogle Scholar
- Doi TS, Marino MW, Takahashi T, Yoshida T, Sakakura T, Old LJ, Obata Y. Absence of tumor necrosis factor rescues RelA-deficient mice from embryonic lethality. Proc Natl Acad Sci U S A. 1999;96:2994–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science. 1996;274:787–9.View ArticlePubMedGoogle Scholar
- Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science. 1998;281:1680–3.View ArticlePubMedGoogle Scholar
- Algül H, Treiber M, Lesina M, Nakhai H, Saur D, Geisler F, Pfeifer A, Paxian S, Schmid RM. Pancreas-specific RelA/p65 truncation increases susceptibility of acini to inflammation-associated cell death following cerulein pancreatitis. J Clin Invest. 2007;117:1490–501.View ArticlePubMedPubMed CentralGoogle Scholar
- Heise N, De Silva NS, Silva K, Carette A, Simonetti G, Pasparakis M, Klein U. Germinal center B cell maintenance and differentiation are controlled by distinct NF-kB transcription factor subunits. J Exp Med. 2014;211:2103–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Weingarten-Gabbay S, Elias-Kirma S, Nir R, Gritsenko AA, Stern-Ginossar N, Yakhini Z, Weinberger A, Segal E. Comparative genetics. Systematic discovery of cap-independent translation sequences in human and viral genomes. Science. 2016;351:240.
- Narayanan R, Klement JF, Ruben SM, Higgins KA, Rosen CA. Identification of a naturally occurring transforming variant of the p65 subunit of NF-kappa B. Science. 1992;256:367–70.View ArticlePubMedGoogle Scholar
- Lyle R, Valleley EM, Sharpe PT, Hewitt JE. An alternatively spliced transcript, p65 delta 2, of the gene encoding the p65 subunit of the transcription factor NF-kappa B. Gene. 1994;138:265–6.View ArticlePubMedGoogle Scholar
- Epelman S, Lavine KJ, Beaudin AE, Sojka DK, Carrero JA, Calderon B, Brija T, Gautier EL, Ivanov S, Satpathy AT, et al. Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation. Immunity. 2014;40:91–104.View ArticlePubMedPubMed CentralGoogle Scholar
- George SH, Gertsenstein M, Vintersten K, Korets-Smith E, Murphy J, Stevens ME, Haigh JJ, Nagy A. Developmental and adult phenotyping directly from mutant embryonic stem cells. Proc Natl Acad Sci U S A. 2007;104:4455–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Farley FW, Soriano P, Steffen LS, Dymecki SM. Widespread recombinase expression using FLPeR (flipper) mice. Genesis. 2000;28:106–10.View ArticlePubMedGoogle Scholar
- de Vries WN, Binns LT, Fancher KS, Dean J, Moore R, Kemler R, Knowles BB. Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis. 2000;26:110–2.View ArticlePubMedGoogle Scholar
- Lewandoski M, Wassarman KM, Martin GR. Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Curr Biol. 1997;7:148–51.View ArticlePubMedGoogle Scholar
- Tieu BC, Ju X, Lee C, Sun H, Lejeune W, Recinos A, Brasier AR, Tilton RG. Aortic adventitial fibroblasts participate in angiotensin-induced vascular wall inflammation and remodeling. J Vasc Res. 2011;48:261–72.View ArticlePubMedGoogle Scholar
- Tieu BC, Lee C, Sun H, Lejeune W, Recinos A, Ju X, Spratt H, Guo DC, Milewicz D, Tilton RG, Brasier AR. An adventitial IL-6/MCP1 amplification loop accelerates macrophage-mediated vascular inflammation leading to aortic dissection in mice. J Clin Invest. 2009;119:3637–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Soriano P, Montgomery C, Geske R, Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991;64:693–702.View ArticlePubMedGoogle Scholar
- Mansour SL, Thomas KR, Capecchi MR. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988;336:348–52.View ArticlePubMedGoogle Scholar
- Xu J. Preparation, culture, and immortalization of mouse embryonic fibroblasts. Curr Protoc Mol Biol. 2005;Chapter 28:Unit 28.21.Google Scholar
- Zhao Y, Widen SG, Jamaluddin M, Tian B, Wood TG, Brasier AR. Quantification of Activated NF-kB/RelA Complexes Using ssDNA Aptamer Affinity–Stable Isotope Dilution-Selective Reaction Monitoring-MS. Mol Cellular Proteomics. 2011;10(6):1–16.View ArticleGoogle Scholar