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Cell Reports, Volume 3, Issue 2, 335-341, 07 February 2013
The ROSA26-iPSC Mouse: A Conditional, Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation
Lieven Haenebalcke1, 2, Steven Goossens1, 2, Pieterjan Dierickx1, 2, Sonia Bartunkova1, 2, Jinke D'Hont1, 2, Katharina Haigh1, 2, Tino Hochepied2, Dagmar Wirth3, Andras Nagy4 and Jody J. Haigh1, 2, ,
1 Vascular Cell Biology Unit, VIB Department for Molecular Biomedical Research, Ghent University, Technologiepark 927, 9052 Zwijnaarde (Ghent), Belgium
2 Department for Biomedical Molecular Biology, Ghent University, Technologiepark 927, 9052 Zwijnaarde (Ghent), Belgium
3 Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
4 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1XG, Canada
Graphical Abstract
► Conditional and inducible reprogrammable mouse model presented ► Allows removal of the Yamanaka factors after reprogramming ► Fully and partially reprogrammed endothelial cells generated ► Lineage-directed differentiation of iPSCs toward beating cardiomyocytes
- Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest—for example, to enhance lineage-directed differentiation—with the use of a trap-coupled RMCE reaction. We show that, similar to ESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes.
Pluripotency can be induced in various cell types by the ectopic expression of four transcription factors Oct4 (also known as Pou5f1), Sox2, Klf4, and c-Myc (hereinafter referred to as OSKM), generating induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). Since the initial discovery in 2006, various methods (González et al., 2011) have been developed to generate iPSCs using inducible or excisable transposon-based strategies to control the ectopic expression of the reprogramming factors instead of the more "classical" and uncontrollable retroviral-based systems. The most advanced techniques in mouse iPSC generation are the recently developed double transgenic reprogrammable mouse systems (Carey et al., 2010; Stadtfeld et al., 2010). These doxycycline-inducible Col1a1 locus-targeted transgenic mouse models allow iPSC derivation from different cell populations when crossed with the ubiquitously expressing reverse-tetracycline-dependent-transactivator (rtTA) ROSA26 mouse strain. However, these double transgenic approaches lack tissue/cell-specific OSKM expression, as well as a means of selecting OSKM-expressing cells, and do not allow the exchange of the reprogramming factors by other genes to study iPSC lineage-directed differentiation at a well-defined locus. Here, we report a conditional and inducible (COIN) transgenic mouse model that allows expression of OSKM exclusively from within the ROSA26 locus. Moreover, after reprogramming, the four factors can be replaced in the resulting iPSCs by any cDNA/ORF of interest via a recombinase-mediated cassette exchange (RMCE) strategy.
Results and Discussion
Generation and Use of the ROSA26-iPSC Mouse Model to Create Reprogrammed Cells
We generated a COIN OSKM construct by using similar conditional strategies (Belteki et al., 2005), as well as previously published (Nyabi et al., 2009) Gateway cloning technologies, and subsequently targeted this construct to the ROSA26 locus via homologous recombination in robust hybrid F1 embryonic stem cells (ESCs), i.e., G4 ESCs (George et al., 2007). In this double-locked system (Figure 1A and Figure S1A), a loxP-flanked (floxed) neomycin resistance (NeoR) transcriptional STOP cassette prevents the expression of the bicistronic rtTA-IRES-puromycin-pA mRNA and the reprogramming factors. Through Cre-mediated deletion of this STOP cassette, rtTA is expressed from the endogenous ROSA26 promoter. Only after administration of the inducing drug doxycycline (Dox), rtTA will undergo nuclear translocation and activate the Tet-responsive element (TRE; tet(o)-CMVmin) that will drive expression of the polycistronic OSKM cassette along with a reporter gene (enhanced green fluorescent protein-Luciferase; EGFP-Luciferase). Via diploid embryo aggregation with correctly targeted ESCs (Figure 1B), a stable mouse line was generated from several strong, germline-transmitting chimeras (Figures S1B and S1C). These ROSA26-iPSC mice can be bred to the wide range of tissue/cell-specific Cre mouse lines available. This allows conditional and inducible expression of the reprogramming factors in the derived primary cell cultures isolated either from embryonic or adult offspring for cellular reprogramming experiments (Figure 2A).
To test our ROSA26-iPSC transgenic model functionally, we isolated mouse embryonic fibroblasts (MEFs) and adult tail tip fibroblasts (TTFs) from Sox2 Cre X ROSA26-iPSC mice. We cultured and expanded these cells under puromycin (Puro) selection before inducing the reprogramming factors with Dox. After 3 weeks of continuous Dox induction, several OSKM-EGFP-expressing colonies with an ESC/iPSC-like morphology could be detected (Figure 2B). Individual picking and expansion of these colonies led to the establishment of multiple Dox-independent iPSC lines expressing pluripotency-related markers such as alkaline phosphatase (Alk. Phos.), Oct4, Nanog, and SSEA1 (Figure 2C) and increased Nanog mRNA expression levels that are similar to wild-type G4 ESCs (Figure 2D). In vitro differentiation of these iPSCs using embryoid body differentiation assays demonstrated no differences compared to wild-type ESCs in terms of the formation of embryoid bodies, beating cardiomyocyes, sprouting vascular endothelial channels, or their hematopoietic differentiation potential (Figure 2E; data not shown). To further characterize the differentiation potential of our iPSC clones in vivo, cells were subcutaneously injected into immunocompromized nude mice, which led to teratoma formation for all analyzed clones. Histological analysis of the tumors demonstrated their differentiation potential toward cell types of the three different germ layers (Figure 2F). Moreover, adult iPSC-derived chimeric mice could be generated using diploid complementation assays, thereby further underscoring the pluripotent nature of these cells (Figure 2G).
However, as previously reported for other TRE-based transgenic mouse lines (Beard et al., 2006; Premsrirut et al., 2011), we also observed significant mosaic transgene expression in our ROSA26-iPSC model. Indeed, of our starting fibroblast populations, only around 8% of the MEFs (OSKM-heterozygous) and 16% of the TTFs (OSKM-homozygous) appeared to be capable of turning on the OSKM-EGFP expression cassette after 2 days of in vitro Dox induction (Figure S2A). As well, when we turned on the reprogramming factors in vivo for 5 days in a hematopoietic/endothelial lineage-specific manner using Tie2 Cre X ROSA26 OSKM-homozygous mice, we could sort, based on the EGFP-based transgene expression of our mouse model, EGFP/c-Kit-positive hematopoietic stem/progenitor cells (HS/PCs) (Figure S2B) and reprogram them toward iPSCs with similar characteristics as described before (data not shown). Therefore, using our model and EGFP-based fluorescence-activated cell sorting (FACS), it is possible to isolate cells either from in vivo sources or after in vitro expansion that are still capable of turning on the OSKM reprogramming factors.
Additionally, it should be noted the reprogramming efficiency in our developed mouse model is somewhat less efficient in generating iPSCs (Document S1. Tables S1 and S2) than the other two previously published Col1a1-based reprogrammable mouse models (Carey et al., 2010; Stadtfeld et al., 2010). To understand the reason for this lower efficiency, we directly compared the OSKM mRNA and protein levels in our ROSA26-iPSC model with those of the model developed by Carey et al., 2010. Upon Dox administration, significantly lower mRNA and protein levels of the reprogramming factors were observed in OSKM-homozygous, EGFP-positive sorted MEFs from our ROSA26-based model compared to OSKM-homozygous MEFs from the Carey et al., 2010 Col1a1-based model (Figures S2C and S2D). The lower frequencies of reprogramming observed in our ROSA26-OSKM-based model can therefore be explained by the overall lower OSKM expression levels. It is interesting that these differences are less pronounced in our ROSA26-based TTF-iPSCs that appear to have basal OSKM levels more similar to wild-type ESCs than in the Col1a1-derived TTF-iPSCs (Figure S2E). As well, unlike the model published by Stadtfeld et al., 2010, we see no evidence of spontaneous tumor formation in adult Sox2 Cre X OSKM-homozygous mice under noninduced conditions up to 18 months of age. This demonstrates the tightness of OSKM expression in our system as also shown by quantitative real-time PCR (qRT-PCR) analysis of derived MEFs compared to wild-type MEFs (Figure S2C).
Not only did we obtain fully reprogrammed iPSCs from our ROSA26-iPSC model, but we also generated interesting partially reprogrammed cells (discussed later) derived from EGFP/CD31-positive endothelial cells (ECs) (Figure S3A). Upon Dox removal, these cells have the potential to differentiate spontaneously back into CD31-positive, vascular-like channels in vitro even without the addition of additional cytokines such as VEGF (Figures S3B and S3C). These cells are somewhat similar to what has recently been described for rat OSKM-induced vascular progenitor cells (iVPCs) (Yin et al., 2012) in their ability to differentiate spontaneously back into CD31-positive cells when Dox was removed from the system even in late passage (p > 10) clones. Of the three iVPC clones analyzed in greater detail, all showed discrete populations of Alk. Phos.-positive cells (Figure S3D), but only clone 3 was capable of forming a tumor when injected into immunocompromised mice. It is interesting that the tumor did not contain a variety of different tissues derived from all three different germ layers characteristic for teratomas but instead displayed a rather limited developmental potential with high vascular channel formation capacity as judged by CD31 immunostaining analysis (Figure S3E). By selectively replating clone 3 on to gelatinized plates and subcloning ESC-like colonies, we could derive Dox-independent EC-derived iPSCs (EC-iPSCs) that ubiquitously expressed ESC cell markers such as Alk. Phos. and Oct4 (Figure 2H). These reprogrammed EC-iPSCs have a greater developmental potential than the original iVPC precursor cells and could form teratomas with all three germ layer derivatives along with a more limited angiogenic potential (Figure S3E). Although not "truly" pluripotent, the iVPCs in comparison to EC-iPSCs can be of great interest in preclinical studies as well as in further unraveling the molecular mechanisms of cellular reprogramming and identifying genetic determinants of EC memory. Using this conditional and inducible ROSA26-iPSC model system with other tissue/cell-restricted Cre lines will also enable researchers to identify other stable partially reprogrammed cell lines similar to our mouse iVPCs, partially described here. This is an important issue as several concerns have recently been raised about the genetic stability (Blasco et al., 2011) and immunogenicity (Zhao et al., 2011) of true pluripotent iPSCs.
Characterization and Validation of the RMCE Component of the ROSA26-iPSC Mouse Model in Lineage-Directed Cellular Differentiation Assays
After successful reprogramming, the ectopic expression of OSKM is no longer needed and can be shut off by withdrawal of Dox. However, we designed our construct in such a way that we can actually use the same ROSA26 locus to remove and, more important, exchange the reprogramming factors using a trap-coupled RMCE reaction with high efficiencies as previously reported (Sandhu et al., 2011). Other cDNAs/ORFs of interest can hereby be efficiently cloned into Gateway-compatible vectors and introduced in a conditional (pRMCE DV1) or conditional and inducible (pCOIN DV) manner (Figure 3A). By cotransfecting such RMCE-compatible incoming vectors together with a FlpE-expressing plasmid, the resulting NeoR iPSC clones should be correctly retargeted and can, for example, subsequently be used for lineage-directed differentiation studies (Figure 3B). To demonstrate this, we created two incoming in vitro Cre-excised lacZ-encoding constructs: one pRMCE DV1 lacZ construct and one pCOIN DV lacZ construct. In separate experiments, we successfully replaced the reprogramming factors by these lacZ constructs. For the pRMCE DV1 lacZ, we obtained constitutive ROSA26-based lacZ expression in the retargeted iPSCs (Figure 3C). Using beta-galactosidase staining analysis, we could show that the RMCE approach was working at high efficiencies in MEF-derived (12/12 lacZ-positive/NeoR clones = 100%), TTF-derived (22/22 lacZ-positive/NeoR clones = 100%), and EC-derived (18/19 lacZ-positive/NeoR clones = 95%) iPSC clones. The exchange was further validated using three randomly chosen NeoR RMCE DV1 lacZ MEF-iPSC clones to demonstrate the complete removal of the COIN OSKM transgene by genomic PCR analysis (Figures S4A and S4B). Moreover, high percentage E11.5 chimeric embryos that constitutively express lacZ could be derived from retargeted TTF-iPSC clones, thereby demonstrating that the pluripotency of these cells is not affected after RMCE with lacZ (Figure 3C). For the pCOIN DV lacZ system, the expression was, as expected, only induced after Dox supplementation, which once again demonstrates the tight regulation of this COIN system (Figure 3D).
To functionally test our system further and specifically differentiate our iPSCs toward beating cardiomyocytes as a promising cell type for preclinical regenerative and tissue repair studies, we successfully exchanged the OSKM factors in MEF-iPSCs by a Cre-excised pCOIN DV construct encoding Mesp1, a known key transcriptional regulator of cardiovascular development (Bondue and Blanpain, 2010). Similar to previously published results in ESCs (Lindsley et al., 2008), a brief 2-day pulse of Mesp1 expression in early differentiating iPSCs (Figure 4A), together with Wnt inhibition (using the Wnt pathway inhibitory compound IWR1; Chen et al., 2009), preferentially generated enhanced numbers of cells positive for the definitive cardiac marker TroponinT (cTnT) (Figure 4B). We also observed significantly increased mRNA expression levels of other cardiac marker genes such as Nkx2.5 as judged by qRT-PCR mRNA expression analysis (Figure 4C; data not shown) and in the number of beating cardiomyocytes (Figure 4D; Movie S1. Random Differentiation of COIN Mesp1 Retargeted MEF-iPSCs, Related to Figure 4 and Movie S2. Lineage-Directed Differentiated of COIN Mesp1 Retargeted MEF-iPSCs, Related to Figure 4).
We believe that the exchange of the reprogramming factors by other genes from within the same ROSA26 locus via an efficient RMCE reaction, together with in vitro validation of the resultant exchanged iPSC clones, will be of great importance to the field of cellular (de)differentiation in efficiently identifying genes that can control cell lineage-directed differentiation. This was demonstrated by the cardiomyocyte-specific differentiation of mouse-derived iPSCs by ectopic Mesp1 expression together with Wnt inhibition. This regulated transgene expression will be particularly useful in preclinical regenerative medicine approaches where continued expression of powerful transcriptional modulators may be detrimental for terminal cellular differentiation necessary for tissue repair.
In summary, we have created an in vivo resource that will add to the toolbox of existing mouse resources that will allow scientists to identify genes more easily and understand their roles in cellular (de)differentiation processes. It should also be noted that, given the discovery of the human counterpart of the ROSA26 locus (Irion et al., 2007) as well as other safe genomic loci (Hockemeyer et al., 2009), our system could be similarly adapted for human cell-based studies and identifying factors involved in human ESC/iPSC (de)differentiation.
Experimental Procedures
Generation of the ROSA26-iPSC Mice
The construction of the expression vector, subsequent ESC targeting/screening, and final generation of ROSA26-iPSC transgenic mice were performed using a similar strategy as described elsewhere (Nyabi et al., 2009). Briefly, the individual constructs were first created by directional cloning strategies of the specific sequences into Gateway-compatible vectors and subsequently recombined using a multisite Gateway LR reaction. PvuI-linearized DNA of the resulting expression vector was then electroporated into wild-type G4 ESCs (George et al., 2007) and grown under standard ESC conditions with G418 selection drug added to the culture medium. Individual G418-resistant ESC colonies were picked, expanded, and screened by PCR and Southern blot analysis. One of the correctly ROSA26-targeted ESC clones was subsequently aggregated with diploid E2.5 Swiss embryos and generated several high-grade, germline-transmitting chimeric animals.
Cellular Reprogramming
(1) Fibroblasts isolated from Sox2 Cre (Lu and Robertson, 2004) X ROSA26-iPSC E13.5 embryos and tail tips of 6-week-old adult mice were first expanded under Puro selection before being plated on to culture dishes coated with a mitomycin C-inactivated feeder layer and reprogrammed in daily refreshed ESC medium or KSR medium (ESC medium with 15% Knockout Serum Replacement [Invitrogen] instead of fetal bovine serum) supplemented with doxycycline hyclate (Dox, 2 μg/ml, Sigma-Aldrich). (2) OSKM-EGFP-expressing HS/PCs were isolated from 3-week-old and in vivo Dox-induced (5 days of daily refreshed Dox in the drinking water, 1 mg/ml) Tie2 Cre (Kisanuki et al., 2001) X ROSA26-iPSC mice. Cells extracted from the bone marrow or spleen were first stained with a c-Kit-specific antibody (APC-H7 rat antimouse CD117 [c-Kit], PharMingen) before double EGFP/c-Kit-positive HS/PCs were sorted on a FACSAria II machine (BD Biosciences) and allowed to reprogram similarly as described for the fibroblasts. (3) Similarly as for the HS/PCs, ECs were isolated from lungs of 3-week-old and in vivo Dox-induced (5 days of daily refreshed Dox in the drinking water, 1 mg/ml) Tie2 Cre (Kisanuki et al., 2001) X ROSA26-iPSC mice. CD31-positive ECs were isolated using magnetic-activated cell sorting (MACS) with incubated CD31 (PECAM, rat antimouse CD31 antibody, BD Biosciences) precoated sheep antirat immunoglobulin G (IgG) magnetic Dynabeads (Dynal, Life Technologies) and reprogrammed similarly as described for the fibroblasts. Initially, only partially reprogrammed individual iVPC colonies could be observed and expanded in the presence of Dox. Subcloning of one particular iVPC clone on gelatinized plates eventually led to Dox-independent EC-iPSCs.
Immunohistochemistry labeling of various different markers were performed using standard procedures and/or commercially available kits according to the manufacturer's instructions: Alk. Phos. (Mouse ESC/iPSC Characterization Set [Stemgent] or Leukocyte Alkaline Phosphatase Kit [Sigma-Aldrich]), Oct4 (Mouse ESC/iPSC Characterization Set [Stemgent] or mouse anti-Oct4 primary antibody (Santa Cruz Biotechnology]), Nanog (Mouse ESC/iPSC Characterization Set [Stemgent]), SSEA1 (Mouse ESC/iPSC Characterization Set [Stemgent]), CD31 (rat anti-CD31 antibody [BD Biosciences]), cTnT (mouse anti-cTnT antibody [NeoMarkers]) and X-gal (previously described in Novak et al., 2000).
Total RNA was isolated using RNeasy Plus Mini Kit (QIAGEN). cDNA was synthesized using the First Strand cDNA Synthesis Kit (Roche) with oligo(dT) primer by starting from equal amounts of RNA as measured by a NanoDrop spectrophotometer (Thermo Scientific). qRT-PCRs were performed using the LightCycler 480 SYBR Green I Master (Roche), monitored on a LightCycler 480 system (Roche), and analyzed using qBase software (Hellemans et al., 2007). Gene expression was standardized against housekeeping genes hydroxymethylbilane synthase (Hmbs), β-actin, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh). All primers are listed in Document S1. Tables S1 and S2. The error bars in the graphs (Figures 2D, 4A, 4C, and S2C) represent the SDs of the average of triplicate reactions (technical replications).
In Vitro Differentiation
(1) Differentiation of ESCs/iPSCs toward hematopoietic cell lineages was performed first by embryoid body (EB) formation in 1% methylcellulose (R&D Systems) followed by replating and further differentiation in Methocult GF3434 medium (Stem Cell Technologies) according to the manufacturer's instructions. Colonies were blindly scored after 12 days of culture. The error bars in the graph (Figure 2E) represent the SD of the average of triplicate biological assays. (2) iPSC differentiation preferentially toward cardiomyocytes was performed as previously described for ESCs (Lindsley et al., 2008). Early differentiating iPSC EBs were plated (1.5 × 105 cells per milliliter) and grown in bacterial grade Petri dishes in EB differentiation medium and supplemented with Dox (2 μg/ml) and/or IWR1 (10 μM, Sigma-Aldrich).
Teratoma Formation
iPSCs were trypsinized and subcutaneously injected into the flank of immunocompromised nude mice. Forming teratomas were isolated 5 to 8 weeks later and further processed to paraffin sections stained with hematoxylin and eosin (H&E) for histological analysis. All experiments performed on mice were approved by the animal ethical committee of Ghent University.
RMCE Targeting
For the trap-coupled RMCE experiments, Effectene transfection reagent (QIAGEN) was used according to the manufacturer's instructions to cotransfect an incoming vector, created via directional cloning steps of individual Gateway-compatible constructs and subsequently recombined using Gateway LR reactions as previously described (Nyabi et al., 2009), together with a FlpE-expressing plasmid (pCAGGS-FlpE-IRES-puromycin-pA) (Schaft et al., 2001) in a 1:1 ratio into RMCE-compatible iPSCs. G418 selection (200 μg/ml) was started 48 hr after transfection. After 7 days, individual NeoR RMCE-retargeted iPSC colonies could be observed and further expanded.
Beating Cardiomyocyte Assay
For each condition, 96 individually plated embryoid bodies were blindly scored from day 8 until day 12 of differentiation for the presence of beating cardiomyocytes.
For further details, please see the Extended Experimental Procedures.
Vector Design and Gateway Cloning
(1) The genes encoding for the reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) were cut out of pPB-CAG.OSKM-puΔt (Yusa et al., 2009) as a EcoRI/ClaI-blunt fragment and ligated into the pENTR1A vector cut by EcoRV and EcoRI, hereby eliminating the ccdB negative selection gene (Katzen, 2007). The pEntry 5′ COIN was created by directional cloning of the rtTA sequence (from the plasmid pSPC-rtTA (Perl et al., 2002)) followed by the IRES-puromycin-pA, INS-INS (tandem of chicken β-globin insulator core sequences (Yusufzai and Felsenfeld, 2004) shown to protect against silencing effects (Sekkali et al., 2008)) and TRE (tet(o)-CMVmin) genetic features into the multisite Gateway-compatible pEntry L4-ccdB-R1 vector. The insulated pEntry 3′ COIN was also created by directional cloning of the IRES-EGFP-Luciferase-pA and INS-INS sequences into the multisite Gateway-compatible pEntry R2-ccdB-L3 vector. The destination vector used to make the stable ROSA26-iPSC mouse line was created as follows: A pBigT R4-ccdB-R3 plasmid, with a functional floxed PGK-neomycine STOP cassette, was digested with PacI/AscI and ligated into a specifically designed EcoRV/MluI-digested synthetic construct (http://www.GenScript.com) harboring the FRT sites and the promoter-ATG-less neomycine-pA. The resulting construct was subsequently digested with PacI/AscI and ligated into the PacI/AscI-digested pROSA26 vector (Soriano, 1999). As described before (Nyabi et al., 2009), a multisite Gateway LR reaction was put in sequentially first by combining the three Entry vectors (pEntry 5′ COIN, pENTR1A OSKM, and insulated pEntry 3′ COIN) followed by an overnight reaction together with the RMCE-compatible pROSA26 COIN vector. Several colonies were obtained after electroporating 5 μl of the LR reaction mixture into DH5α bacteria that were subsequently plated onto ampicillin-containing bacterial plates. (2) For the construction of the pRMCE DV1, we first digested our previously modified pBigT R1-ccdB-R2 IRES-EGFP-pA construct (Nyabi et al., 2009) with StuI/EcoRV followed by self-ligation of the 7.8 kb vector backbone, deleting most of the PGK-neomycin resistance sequence. This pBigT delta-Neo R1-ccdB-R2 IRES-EGFP-pA was then cut as a PacI/AscI fragment and ligated into a specifically designed synthetic construct ordered from GenScript (http://www.GenScript.com), which has all the genetic features (wt FRT, PacI-AscI, INS-INS, PGK-ATG-F5 FRT) to allow a trap-coupled RMCE reaction. The resulting pRMCE DV1 was subsequently used in a monosite Gateway LR reaction as previously described (Nyabi et al., 2009) together with a pENTR1A lacZ construct created by the XbaI digestion of the pBI-3 (Baron et al., 1995) and ligation of the nls-lacZ sequence into the pENTR1A. The pCOIN DV was created using a similar strategy. We created a similar pBigT delta-neo R4-ccdB-R3 construct whereby most of the PGK-neomycin resistance gene was deleted by StuI/EcoRV digest and self-ligation of the 6.6 kb pBigT R4-ccdB-R3 vector backbone. PacI/AscI digestion of that vector, followed by ligation into the same specifically designed synthetic construct used for creating pRMCE DV1 and also cut with PacI/AscI, allowed us to generate the pCOIN DV. The same pEntry 5′ COIN and similar pEntry 3′ COIN (this time without the INS-INS sequences as already present in the pCOIN DV) together with the pENTR1A lacZ or the kindly provided pENTR1A Mesp1 construct (by A. Bondue and C. Blanpain, ULB, Belgium) were used in separate (multisite) Gateway LR reactions to generate pCOIN DV cDNA (lacZ/Mesp1) targeting constructs to be used in trap-coupled RMCE reactions. (3) Both pRMCE DV1 and pCOIN DV were also in vitro Cre-excised by incubating these plasmids for 1 h at 37°C with recombinant Cre protein (VIB-DMBR protein core facility) followed by 5 min at 70°C to inactivate the recombinant Cre protein. These resulting Cre-excised plasmids were just as efficiently used in Gateway LR reactions compared to the non-Cre-excised versions described above. All vectors described were confirmed by restriction enzyme digests and sequencing analysis.
ESC Targeting via Homologous Recombination and Screening
Twenty micrograms of the PvuI-linearized RMCE-compatible pROSA26 COIN OSKM expression construct was electroporated into wild-type G4 ESCs (George et al., 2007) grown on mitomycin C-treated feeder layer and incubated at 37°C with 5% CO2 and 95% humidity. After 24 hr, G418 (200 μg/ml) was added to the ESC medium (Knockout DMEM (Invitrogen) supplemented with 15% FBS (Hyclone), 0.1 mM β-mercaptoethanol, 2 mM L-glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), penicillin (100 U/ml)-streptomycin (100 μg/ml) (Invitrogen) and 2000 U/ml leukemia inhibitory factor (VIB-DMBR protein core facility). After 7-10 days, 96 G418-resistant colonies were picked and analyzed by PCR (targeted allele: 1.3 kb; primers G1 5′-TAG GTA GGG GAT CGG GAC TCT-3′ and G2 5′-GCG AAG AGT TTG TCC TCA ACC-3′, see also Figure S2A) and Southern blotting (5′ probe, BamHI; EGFP internal probe, KpnI; 3′ probe, KpnI) similar as previously described (Nyabi et al., 2009). Correctly targeted ESC clones were then further expanded and used for generating mouse chimeras.
Diploid Aggregation and PCR Genotyping
(1) Briefly, embryos were collected at day E2.5 from superovulated Swiss female mice. Zonae pellucida of embryos were removed by treatment with acid Tyrode's solution (Sigma-Aldrich). ESC colonies were briefly treated with 0.25% trypsin-EDTA (Invitrogen) to form loosely connected clumps of 7–10 cells. Each zona-free embryo was aggregated with a clump of 7–10 cells inside depression wells made in a 35-mm plastic dish (VWR International) with an aggregation needle (BLS Ltd, Hungary) and cultured overnight in microdrops of KSOM with amino acids (Biognost) under mineral oil (Sigma-Aldrich) at 37°C with 5% CO2 and 95% humidity. The next day, blastocysts were transferred into the uteri of 2.5 days postcoitum (dpc) pseudopregnant Swiss females previously mated with vasectomized males. Chimeras were identified at birth by the presence of black eyes and later by agouti coat pigmentation. Chimeric males with 100% coat color contribution were individually bred with C57BL/6J females. All experiments performed on mice were approved by the animal ethical committee of Ghent University. (2) PCR genotyping of the ROSA26-iPSC mice can be performed by doing a ROSA26 PCR (wild-type allele: 600 bp, mutant allele: 360 bp; primers ROSA26 fw 5′-AAA GTC GCT CTG AGT TGT TAT-3′, ROSA26 rev wt 5′-GGA GCG GGA GAA ATG GAT ATG-3′ and ROSA26 rev mut 5′- GCG AAG AGT TTG TCC TCA ACC-3′) and an OSKM transgene-specific PCR (transgene: 120 bp; primers OSKM fw 5′-GGC TCT CCC ATG CAT TCA AAC G-3′ and OSKM rev 5′-GCT TCA GCT CCG TCT CCA TCA TGT-3′). For details, see Figure S4A.
MEFs-TTFs-HS/PCs-ECs Isolation and Reprogramming
(1) Mouse embryonic fibroblasts (MEFs) were isolated by sacrificing Sox2 Cre (Lu and Robertson, 2004) X OSKM-heterozygous embryos at E13.5 removing the head and internal organs. The remaining tissues were then physically dissociated and treated with 0.25% trypsin-EDTA (Invitrogen) at 37°C for 20 min. After passing the tissue/cells several times through a 2 ml syringe with a 20 gauge needle, the cell mixture was resuspended in MEF medium (DMEM (Invitrogen) supplemented with 15% FBS (Hyclone), 0.1 mM β-mercaptoethanol, 2 mM L-glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), penicillin (100 U/ml)-streptomycin (100 μg/ml) (Invitrogen) and 1 mM sodium pyruvate (Invitrogen)). After 24 hr, puromycin (Puro, 1.25 μg/ml) was added to the culture medium to select for Cre-excised, rtTA-expressing MEFs. (2) Tail tip fibroblasts (TTFs) were isolated from 6 weeks old, adult Sox2 Cre X OSKM-homozygous mice by culturing skin/hair-stripped pieces of tail in a minimal amount of MEF medium. Fibroblasts were allowed to grow out of these tail tips and nearly confluent TTF cell cultures were further expanded and put under puromycin selection (1.25 μg/ml) to select for Cre-excised, rtTA-expressing TTFs. (3) For the reprogramming of fibroblasts (both MEFs as well as TTFs) PuroR fibroblasts were plated unto culture dishes (∼3000 cells/cm2) coated with a mitomycin C-inactivated feeder layer and grown in ESC or KSR medium ( = ESC medium with 15% Knockout Serum Replacement (Invitrogen) instead of FBS) supplemented with doxycycline hyclate (Dox, 2 μg/ml, Sigma-Aldrich). The medium, containing Dox, was refreshed every day while nuclear reprogramming took place. After 3 weeks of Dox treatment, individual EGFP-expressing colonies were picked and further expanded in normal ESC medium on inactivated feeders. (4) For hematopoietic stem/progenitor cells (HS/PCs), 5 days prior to the isolation and reprogramming of these cells, 3-week-old Tie2 Cre (Kisanuki et al., 2001) X OSKM-homozygous mice were given Dox via their drinking water (1 mg/ml). After those 5 days, bone marrow was flushed or the spleen was treated with a collagenase A solution (0.2% collagenase in PBS supplemented with 10% FBS) for 1 hr at 37°C to harvest HS/PCs. The cells were stained with an APC-coupled c-Kit specific antibody (APC-H7 rat antimouse CD117 (c-Kit), PharMingen) before double EGFP/c-Kit-positive HS/PCs were sorted on a FACSAria II machine (BD Biosciences) and immediately plated unto a six-well plate (500 cells/well) coated with inactivated feeder cells and grown in ESC medium supplemented with 2 μg/ml Dox. After 3-6 weeks of daily refreshing of the medium, individual Dox-independent HS/PC-iPSCs could be observed and expanded. (5) For mouse endothelial cells (ECs), 5 days prior to the isolation and reprogramming of these cells, 3-week-old Tie2 Cre X OSKM-homozygous mice were given Dox via their drinking water (1 mg/ml). After those 5 days, the lungs of these mice were isolated and treated with a collagenase A solution (0.2% collagenase in PBS supplemented with 10% FBS) for 1 h at 37°C. CD31-positive ECs were isolated from the cell suspension using magnetic-activated cell sorting (MACS) with incubated CD31 (PECAM, rat antimouse CD31 antibody, BD Biosciences) precoated sheep antirat IgG magnetic Dynabeads (Dynal, Life Technologies). The CD31-enriched cell population was immediately plated onto a 10 cm dish coated with inactivated feeder cells and grown in ESC cell medium supplemented with 2 μg/ml Dox. After 3-6 weeks of daily refreshing of the medium, induced vascular progenitor cells (iVPCs) could be observed and expanded. Withdrawal of the Dox from the medium allowed the iVPCs to differentiate into vascular-like, CD31-positive structures within 2 weeks in feeder-coated dishes. Subcloning of iVPC clone 3 on gelatinized plates eventually led to Dox-independent EC-iPSCs. (6) Reprogramming efficiencies were calculated based on the amount of expanded Dox-independent ESC/iPSC-like colonies compared to the total amount of cells capable of expressing the OSKM-EGFP transgene plated.
(1) ESCs and iPSCs were analyzed for pluripotency markers (alkaline phosphatase [Alk. Phos.], Oct4, Nanog, and SSEA1) using a Mouse ESC/iPSC Characterization Set (Stemgent). The immunofluorescence analysis was performed according to the manufacturer's instructions. Alternatively, iPSCs were stained for alkaline phosphatase activity using the Leukocyte Alkaline Phosphatase Kit (Sigma-Aldrich) according to manufacturer's recommendations. Additional Oct4 stainings were performed fixing iPSCs in 4% paraformaldehyde (PFA), permeabilizing with 0.25% TX-100 and incubated overnight at 4°C with mouse anti-Oct4 primary antibody (1:100; C-10 Santa Cruz Biotechnology). Next, the cells were incubated overnight at 4°C with DyLight 488 antimouse IgG secondary antibody (1:1,000; Thermo Scientific). Nuclei were stained with DAPI (1:1,000; Molecular Probes, Invitrogen). (2) The sprouting vasculature from induced vascular progenitor cells (iVPCs) was monitored with CD31 (PECAM) immunofluorescence. Briefly, cells were fixed in 4% PFA and stained with rat antimouse CD31 antibody (1:500; BD Biosciences) followed by incubation with Alexa 568 antirat antibody (1:250; Molecular Probes, Invitrogen). (3) For the cardiac TroponinT (cTnT) immunohistochemistry staining, embryoid bodies were plated at day 4 of differentiation onto Collagen I-coated slides (BD Biosciences) and on day 8 fixed in 2% PFA in PBS for 15 min, washed three times with PBS, and blocked with 1% BSA/0.1% Triton in PBS. Primary antibody anticardiac isoform of TroponinT Ab1 (1:100; NeoMarkers, Fremont, CA; clone 13-11 mouse monoclonal) was added in blocking solution and left overnight at 4°C. Primary antibody was revealed with the secondary antibody Alexa 568 antimouse IgG (Molecular Probes, Invitrogen). Nuclei were stained with Hoechst 33342 (1 μg/ml, Molecular Probes, Invitrogen).
In Vitro Differentiation Assays
Before starting with embryoid body (EB) formation and hematopoietic differentiation, ESCs/iPSCs were feeder depleted and grown for 24 hr on gelatinized plates. For the in vitro hematopoietic differentiation of ESCs/iPSCs, embryoid bodies were generated in 1% methylcellulose (R&D Systems) in EB differentiation medium (IMDM medium (Invitrogen), 15% FBS (Hyclone), 5% PFMHII (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.4 mM MTG (Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma-Aldrich)) supplemented with 300 μg/ml transferrin (Sigma-Aldrich), 100 ng/ml SCL (Stem Cell Technologies), 25 ng/ml IL-11 (Stem Cell Technologies) and 1 ng/ml IL-3 (Stem Cell Technologies). After 10 days of differentiation, EBs were harvested, dissociated with collagenase/DNase at 37°C for 1 hr. Single cells were replated at a concentration of 5 × 104 cells/ml in Methocult GF3434 (Stem Cell Technologies) according to the manufacturer's instructions.
Flow Cytometric Analysis
(1) Cre-excised ROSA26-iPSC mouse-derived fibroblasts were grown and expanded for 7-10 days in MEF medium under Puro selection (1.25 μg/ml) before they were induced for 2 days with 2 μg/ml Dox and profiled on a FACSCalibur machine (BD Biosciences). Gates were set using non-induced control fibroblast cultures as well as eliminating cell debris. (2) Mouse endothelial cells (ECs) were isolated as described above. The CD31-enriched cell population was plated and expanded in EC medium (MEM-α medium (Invitrogen) supplemented with 15% FBS (Hyclone), 0.1 mM β-mercaptoethanol, 2 mM L-glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), penicillin (100 U/ml)-streptomycin (100 μg/ml) (Invitrogen), 1 mM sodium pyruvate (Invitrogen), heparin (3 U/ml, Sigma) and ECGS (14 μg/ml, Biomedical Technologies) for 2 passages before they were induced for 2 days with 2 μg/ml Dox. After a CD31-APC staining (APC rat antimouse CD31, PharMingen), these cells were profiled on a FACSCalibur machine (BD Biosciences). Gates were set using unstained control MEC cultures as well as eliminating cell debris.
Western Blotting
Protein lysates of OSKM-homozygous noninduced and induced (2 days on 2 μg/ml Dox) ROSA26-iPSC MEFs (EGFP sorted; FACSAria II/III machine; BD Biosciences), Col1a1 OSKM MEFs, ROSA26 TTF-iPSCs, Col1a1 TTF-iPSCs and wild-type G4 ESCs were collected in Laemmli buffer (62.5 mM Tris-HCl, pH 6.8; 10% glycerol; 2% SDS and 5% β-mercaptoethanol) containing phosphatase and protease inhibitors (Roche). The total protein concentration was determined using DC Protein Assay (Bio-Rad) according to manufacturer's instructions and 30 μg of each sample was used for detection. Primary antibodies used: mouse anti-Oct4 (1:200; C-10 Santa Cruz Biotechnology), rabbit anti-Sox2 (1:1,000; #2748, Cell Signaling), goat anti-Klf4 (1:1,000; AF3158, R&D Biosystems), rabbit anti-c-Myc (1:1,000; #9402, Cell Signaling) and mouse antiactin (1:10,000; clone C4, Molecular Probes, Invitrogen). Antimouse, antirabbit, or antigoat IgG-HRP secondary antibodies (1:8,000; Sigma-Aldrich) and the ECL detection kit (GE Healthcare) were used for detection.
RMCE Targeting
Correct RMCE-mediated DV1 lacZ targeting was confirmed by PCR (ROSA26 (see above), OSKM (see above), ROSA26-rtTA (fw 5′-AAA GTC GCT CTG AGT TGT TAT-3′ and rev 5′-GGC CGT GTA TCT GAA TGT-3′) and ROSA26-lacZ (fw 5′- AAA GTC GCT CTG AGT TGT TAT-3′ and rev 5′-TGT GCT GCA AGG CGA TTA-3′). For details, see Figure S4A.
X-gal Staining
(1) For X-gal staining on E11.5 embryos, we fixed the tissues for 1 hr in 2% PFA. The embryos were then washed several times in PBS, containing 0.1% sodium deoxycholate and 0.2% Nonidet-P40, at 4°C before put in X-gal staining solution (0.5 mg/ml X-gal, 5 mM potassium ferrocyanide, and 5 mM potassium ferricyanide in wash buffer) overnight at 30°C, protected from light. (2) X-gal staining on cells was performed as previously described (Novak et al., 2000). Briefly, iPSCs were fixed for 10 min in 0.2% glutaraldehyde and washed for 10 min in X-gal wash buffer (2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet-P40 in 100 mM sodium phosphate pH 7.3 or PBS). Subsequently, the cells were stained in X-gal staining solution (0.5 mg/ml X-gal, 5 mM potassium ferrocyanide, and 5 mM potassium ferricyanide in X-gal wash buffer) for 1 hr at 37°C, protected from light.
We thank A. Bradley from the Wellcome Trust Sanger Institute for providing us with the polycistronic OSKM construct, G. Berx from the VIB Department for Molecular Biomedical Research (VIB-DMBR), University of Ghent, for the EGFP-Luciferase reporter construct, and C. Blanpain and A. Bondue from the Free University of Brussels for the Mesp1 construct. We also thank our VIB-DMBR microscopy core facility for their help with the microscopy analysis, K. Deswarte from B. Lambrecht's group for his help with the sorting experiments, and T. Pieters from Professor Frans Van Roy's group (VIB-DMBR) for providing us the Oct4 antibody. L.H. was funded by a Ph.D. grant of the Agency for Innovation by Science and Technology. S.G. and P.D. were supported by the Fonds voor Wetenschappelijk Onderzoek–Vlaanderen.
L.H. and J.J.H. designed the research; L.H. performed the research with help from S.G., P.D., S.B., and J.J.H.; J.D., K.H., and T.H. performed aggregations; L.H. and J.J.H. analyzed the data; A.N. and D.W. provided essential reagents and ideas; L.H. wrote the manuscript together with S.G. and J.J.H.
Supplemental Information
- Movie S1. Random Differentiation of COIN Mesp1 Retargeted MEF-iPSCs, Related to Figure 4 (MOV 709 kb)
- Movie shot at day 8 of randomly differentiated COIN Mesp1 retargeted MEF-iPSCs without pulsed Mesp1 expression or Wnt inhibition (−Dox/−IWR1). No obviously contracting cells can be detected.
- Movie S2. Lineage-Directed Differentiated of COIN Mesp1 Retargeted MEF-iPSCs, Related to Figure 4 (MOV 1700 kb)
- Movie shot at day 8 of differentiated COIN Mesp1 retargeted MEF-iPSCs with pulsed Mesp1 expression (from day 2 until day 4, + Dox) and Wnt inhibition (from day 2 until day 6, + IWR1) enhancing for lineage-directed differentiation toward beating cardiomyocytes. A large proportion of the cells are contracting.
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Publication Information
Received: June 12, 2012
Revised: December 3, 2012
Accepted: January 14, 2013
Published: February 7, 2013


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Bridging the Gaps between Synapses, Circuits, and Behavior
Bridging the Gaps between Synapses, Circuits, and Behavior
Chemistry and Biology, Volume 17, Issue 6,
Pages 607-615,
Publication Date 25 June 2010
Copyright © 2010 Elsevier Ltd All rights reserved.
DOI: 10.1016/j.chembiol.2010.06.001
Pamela M. England
Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
Corresponding author
The decade of the brain may have come and gone, but the final frontier, cracking the neuronal code, still lies ahead. Today, new technologies that allow precise spatiotemporal remote control over the activity of genetically defined populations of neurons within intact neural circuits are providing a means of obtaining a functional wiring diagram of the mammalian brain, bringing us one step closer to understanding precisely how neuronal activity codes for perception, thought, emotion, and action. These technologies and the design principles underlying them are reviewed herein.


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jill werling quinn July 9, 2013 at 01:00 pm
Excuse my rush to get info out to the public. As said above I am trying to place all students into…Read More welcoming homes as quickly as I can, so there may be a slip or two in my grammar when finishing one of my many attempts to get information out to the public about arriving students. If you wish to work on the students grammar when they arrive you may do so. Feel free to pass on my info to others about what I am trying to do.
Heather Kline July 9, 2013 at 01:19 pm
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Quick Facts
- NAME: St. Nicholas
- OCCUPATION: Saint
- BIRTH DATE: c. 280
- DEATH DATE: December 06, 343
- PLACE OF BIRTH: Patara, Turkey
- PLACE OF DEATH: Myra, Turkey
- Full Name: St. Nicholas of Myrna
- AKA: Saint Nick
- AKA: St. Nicholas
- AKA: St. Nick
- AKA: Nikolaos of Myra
- AKA: St. Nicholas of Myra
Best Known For
St. Nicholas was a Christian bishop who provided for the poor and sick, and is the basis for the popular character of Santa Claus.
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Born in Patara, a land that is part of present-day Turkey, circa 280, St. Nicholas was a Christian bishop who helped the needy. After his death, the legend of his gift-giving grew. St. Nicholas transformed into the legendary character called Santa Claus, who brings Christmas presents to children around the world.
Early Life
St. Nicholas was born sometime circa 280 in Patara, Lycia, an area that is part of present-day Turkey. He lost both of his parents as a young man and reportedly used his inheritance to help the poor and sick. A devout Christian, he later served as bishop of Myra, a city that is now called Demre.
There are many legends about St. Nicholas of Myra. One story tells how he helped three poor sisters. Their father did not have enough money to pay their dowries and thought of selling them into servitude. Three times, St. Nicholas secretly went to their house at night and put a bag of money inside. The man used the money so that one of his daughters could marry. On the third visit, the man saw St. Nicholas and thanked him for his kindness. He also reportedly saved three men who were falsely imprisoned and sentenced to death.
Death and Legacy
Several sources state St. Nicholas is believed to have died on December 6, 343. Over the years, stories of his miracles and work for the poor spread to other parts of the world. He became known as the protector of children and sailors and was associated with gift-giving. He was a popular saint in Europe until the time of the Reformation in the 1500s, a religious movement that led to the creation of Protestantism, which turned away from the practice of honoring saints. St. Nicholas, however, remained an important figure in Holland.
The Dutch continued to celebrate the feast day of St. Nicholas, December 6. It was a common practice for children to put out their shoes the night before. In the morning, they would discover the gifts that St. Nicholas had left there for them. Dutch immigrants brought St. Nicholas, known to them as Sint Nikolaas or by his nickname Sinter Klaas, and his gift-giving ways to America in the 1700s.
In America, St. Nicholas went through many transformations and eventually Sinter Klaas became Santa Claus. Instead of giving gifts on December 6, he became a part of the Christmas holiday. In the 1820 poem "An Account of a Visit from St. Nicholas" by Clement Clarke Moore, he is described as a jolly, heavy man who comes down the chimney to leave presents for deserving children and drives a sleigh pulled by flying reindeer. The cartoonist Thomas Nast added to the St. Nicholas legend with an 1881 drawing of Santa as wearing a red suit with white fur trim. Once a kind, charitable bishop, St. Nicholas had become the Santa Claus we know today.
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I still regard Alex Jones and his followers as a bit out there but ´Prison Planet´ is becoming more apt every day.The 21st century police mentality is as oppressive as it is misguided. The presumption of innocence has vanished. Untold hours of training are spent on ´potential threats´ yet we rarely see such a potential threat subdued. We do, however, see ´public servants´ armed to the teeth and glowering at the law-abiding in everyday situations. The S in SWAT used to stand for Special but these paramilitary types can be found everywhere, especially at sporting events, for no good reason.Making matters worse, ideologues have carved out exemptions for certain groups based on skin color, language, dress, etc. Political correctness as practiced by law enforcement is doubly evil since it preys on the innocent and ignores the guilty. Click Here if you Like this Comment
Bloomberg, The Brady gun control weenies, and government officials in Chicago all come from the same hypocritical mindset.YOU cannot be trusted with guns, but they need their protection. It´s elitist, statist tyranny, pure and simple.So-called "shall issue" states are the same. People with money and power as well as celebrities are considered worthy of self-protection. But you plebes and drones that only work to pay taxes are expendable resources to be sacrificed on the altar of "safety".If Costas is scared of guns or cannot trust himself to not shoot some "child" over his loud music, I´m perfectly fine with him not owning any guns. However, he gets armed protection and then wants to deny my family and me OUR protection to salve his twisted, sanctimonius morality. Click Here if you Like this Comment
Costas takes himself and the play games he covers MUCH too seriously. With his somber, dramatic manner one would think he was covering events equal to the WWII Normandy Invasion. Click Here if you Like this Comment 1 person like this.
Costas...a Lib Hype-O-Critter. Click Here if you Like this Comment 1 person like this.
Rosie O´Donut redux. Click Here if you Like this Comment
Why because he is dumb? Click Here if you Like this Comment 1 person like this.
What people have overlooked in the Costas rant is the breaking news story that Nicole Brown Simpson is still alive. According to Costas, were it not for guns, Belcher and his girlfriend would still be alive, so... Click Here if you Like this Comment 1 person like this.
It is an excuse to foment an agenda. Had the lunatic used a knife, they would just be waiting for the next opportunity. Click Here if you Like this Comment
I saw this dullard going on and on and on praising this killer and some of the players wearing black sashes or whatever. They were told to lose them and Costas was a mess. That vicious black nfl man killed his child´s Mom and should have had his body stuffed in a garbage truck along with costas. That poor baby will now be given to gawd knows who. Should never have been mentioned at a game. Click Here if you Like this Comment 1 person like this.
you peons just don´t understand. These people are IMPORTANT... Click Here if you Like this Comment
#2 I think you mean "May Issue" states such as Kalifornia or NY. Shall issue states don´t have a choice if there is no criminal reason for you not to have a carry license. BUT, the whole idea of a "license" to carry or to own for that matter is in violation of the Constitution. The government has NO authority to tell you that you can´t own a gun, and they didn´t until the 1930´s. Click Here if you Like this Comment
This murder could have been prevented by one more gun --- in the hands of the female victim.The 225lb professional linebacker didn´t need a gun; he could have killed her with his bare hands.She, on the other hand, would have only had a chance if she had a gun and knew how to use it. Click Here if you Like this Comment
#11 - I DID mean "may issue" Thanks for catching and pointing out the mistake so others know what I meant. Click Here if you Like this Comment 1 person like this.
Right now Limbaugh is making a well crafted tougue-in-check point by saying this whole thing is not Bob Costas´ fault - - it´s the microphone´s! He´s saying this would never have been heard by anyone if the microphone had been turned off. Afterall, the microphone can cause people person pain and suffering by spreading the words of the wrong people. He´s proposing that microphones need to be regulated. HA HA!Girl´s Hubby Click Here if you Like this Comment
All the ranting against guns by the NFL, and the thing is, they totally have ignored the "Roid Rage" that is now a constant display even on the filed, as players aggressively and feloniously assault each other with vicious deliberate body stomps and head kicks in "field scuffles". The most deadly plague of steroid use infuses the NFL with their super-men players. Congress blatantly leaves the money flush NFL alone on this issue- we love looking at and watching the body jarring concussion riddles play. But, guns are a danger? Idiots and flim-flam- that´s all the NFL is! Like WWE "wrestling" now. Click Here if you Like this Comment