Stage-Specific Conditional Mutagenesis in Mouse Embryonic Stem Cell-Derived Neur - 《stemcells》干细胞学杂志干细胞之家



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江边孤钓

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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Simone Haupta,b, Frank Edenhofera,b, Michael Peitza,b, Anke Leinhaasa, Oliver Brstlea 3 @  Z- L& E9 w0 |$ |+ k
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      【摘要】& w! M7 ?2 n* T; w  k
   Conditional mutagenesis using Cre/loxP recombination is a powerful tool to investigate genes involved in neural development and function. However, the efficient delivery of biologically active Cre recombinase to neural cells, particularly to postmitotic neurons, represents a limiting factor. In this study, we devised a protocol enabling highly efficient conditional mutagenesis in ESC-derived neural progeny. Using a stepwise in vitro differentiation paradigm, we demonstrate that recombinant cell-permeable Cre protein can be used to efficiently induce recombination at defined stages of neural differentiation. Recombination rates of more than 90% were achieved in multipotent pan-neural and glial precursors derived from the Z/EG reporter mouse ESC line, in which Cre recombination activates enhanced green fluorescent proteinexpression. Recombined precursor cells displayed a normal phenotype and were able to differentiate into neurons and/or glial cells, indicating that Cre treatment has no overt side effects on proliferation and neural differentiation. Our data further demonstrate that recombination via Cre protein transduction is not restricted to dividing cells but can even be applied to postmitotic neurons. The ability to conduct Cre/loxP recombination at defined stages of stem cell differentiation in an expression-independent manner provides new prospects for studying the role of individual genes under stringent temporal control.
      【关键词】 Site-specific recombination Neural differentiation Protein transduction Neural repair Stem cell therapy Conditional mutagenesis
   INTRODUCTION
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ESCs represent both a potential source of transplantable cells for regenerative medicine and an excellent model to study development. Over the last decade, ESCs harboring conditional alleles have proven to be very useful in deciphering the role of genes in stem cell self-renewal, embryonic development, and differentiation. In particular, the site-specific recombinase Cre has been used to induce conditional genetic alterations by recombination of previously integrated recombination recognition sites, designated as loxP sites. This 34-base pair recognition site exhibits an overall structure of two inverted repeats and an eight-base pair core or spacer region conferring the directionality of the recombination site. Depending on the relative orientation of the loxP sites with respect to one another, the recombination reaction can result in deletion, inversion, insertion, or translocation of chromosomal DNA with high fidelity ). However, the construction of viral vectors is time consuming and associated with safety concerns. Furthermore, every gene transfer method carries the risk of insertional mutagenesis, which might interfere with other gene functions and hamper the therapeutic use of ESC-derived donor cells. Thus, the traditional modes of delivering Cre represent limiting parameters for the conditional mutagenesis of ESCs and their differentiated progeny.( i9 i6 @: q% v
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In this study, we explored a new strategy for the conditional mutagenesis of ESC-derived neural progeny. This strategy combines growth factor-controlled neural differentiation and direct delivery of Cre recombinase by protein transduction. Protein transduction is a recently developed method to introduce biologically active proteins directly into mammalian cells with high efficiency (for a review, see . Our study was aimed at evaluating the potency of Cre protein transduction to manipulate mouse ESCs and ESC-derived progeny during distinct stages of neural differentiation. Our data demonstrate that direct delivery of biologically active Cre protein can be achieved efficiently and precisely at any stage ranging from pluripotent ESCs to postmitotic neurons. This approach should provide a useful tool for precise control over gene mutation during neural differentiation in vitro." G, C4 [1 E: V% [
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MATERIALS AND METHODS
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ESC Culture and Neural In Vitro Differentiation
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Z/EG ESCs .
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Expression and Purification of Recombinant His-TAT-NLS-Cre Protein2 a% A# M- g- n- V5 G

His-TAT-NLS-Cre (HTNCre) fusion protein was expressed and purified as described . In brief, pTriEx-HTNC plasmid was used to transform Escherichia coli strain TUNER (DE3)pLacI (Novagen, Madison, WI, http://www.merckbiosciences.co.uk/home.asp), allowing isopropyl ß-D-thiogalactoside-inducible expression of His-tagged protein. Cells were harvested by centrifugation and stored at ¨C20¡ãC. Frozen cell pellets were resuspended in lysis buffer (50 mM Na2HPO4 and 5 mM Tris, pH 7.8) at 10 ml/l expression culture. One mg/ml lysozyme and 25 U/ml benzonase (Novagen) were sequentially added to the suspension and incubated for 20 minutes. After a 2-minute sonication step, the lysate was mixed 1:1 with a buffer containing 2 M disodium L-tartaric acid, 20 mM imidazole, 50 mM Na2HPO4, and 5 mM Tris, pH 7.8. The lysate was cleared by centrifugation for 30 minutes at 16,000 rpm at 4¡ãC. Two ml of 50% nickel-nitrilotriacetic acid slurry (Qiagen, Hilden, Germany, http://www1.qiagen.com) was added to the cleared lysate of 1 liter of expression culture and mixed gently at 4¡ãC for 1 hour. The slurry was packed into a column and washed with 10 bed volumes of washing buffer (15 mM imidazole, 500 mM NaCl, 50 mM Na2HPO4, and 5 mM Tris, pH 7.8). Cre protein was eluted with 3 bed volumes of elution buffer (250 mM imidazole, 500 mM NaCl, 50 mM Na2HPO4, and 5 mM Tris, pH 7.8) and dialyzed against either the appropriate cell culture media (see below) or a glycerol buffer (50% glycerol, 500 mM NaCl, and 20 mM HEPES). Protein concentrations were measured using Bradford reagent (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) with a bovine serum albumin standard.2 t6 u. x5 k/ ]" ^
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Cre Protein Transduction. m4 e& m' c( X+ f, y4 y

Prior to Cre transduction, Z/EG cells were plated on appropriate plates containing mitomycin C-treated embryonic feeder cells. Six hours after plating, ESC medium was changed to medium containing the indicated concentrations of purified HTNCre and incubated for the indicated periods of time. After transduction, cells were washed once with phosphate-buffered saline (PBS) and further cultivated for 48 hours in normal medium prior to further analysis. Protein transduction into neural precursor cells was carried out as follows: prior to Cre transduction, pan-neural or glial precursors were cultured for at least 4 days in DMEM/Ham's F-12 medium supplemented with the appropriate growth factors. After plating 1 x 105 cells per cm2 from a single-cell suspension of plated EBs cultured for 5¨C6 days in ITSFn medium or after plating 8 x 104 glial precursor cells per cm2 on PO-coated cell culture dishes, the cells typically reached 70% confluence after 4 days. For Cre transduction, Cre protein was diluted in N3 medium and sterilized by filtration using a 0.2-µm filter. Final concentration of Cre protein in N3 medium after sterilization was determined using Bradford reagent. Pan-neural and glial precursors were incubated with N3 medium containing Cre protein and 10 ng/ml FGF-2 or 10 ng/ml FGF-2 and 20 ng/ml EGF for the indicated times. For Cre transduction of postmitotic neurons, pan-neural precursors plated at 8 x 104 cells per cm2 were cultured for 4 days in the presence of 10 ng/ml FGF-2. After subsequent growth factor withdrawal for another 4 days, cells were incubated for 22 hours in N3 medium containing 1.5 µM Cre protein and 10 µM bromodeoxyuridine (BrdU) for the identification of postmitotic neurons. After transduction, HTNCre-containing medium was replaced by BrdU-supplemented medium for another 26 hours. Cells were fixed with 4% paraformaldehyde and washed three times with PBS, permeabilized with 0.5% Triton X-100 in 1x PBS for 30 minutes, washed once in PBS and incubated with 2 N HCl for 10 minutes. Cells were washed two times with PBS and equilibrated in 0.1 M sodium borate for 10 minutes. The cells were then pretreated with 10% normal goat serum and 1% Triton X-100 in PBS for 30 minutes and subsequently incubated with a monoclonal antibody against BrdU.# ^4 P+ \; a8 u9 N& N
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Southern Blot Analysis, Flow Cytometry, and 5-Bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal) Staining

For Southern blot analysis, Cre-treated cells were harvested 48 hours after transduction. Genomic DNA was extracted from cells by proteinase K digestion and isopropanol precipitation as described previously .

Immunofluorescence# B& H3 |% W9 a; g7 Q

Pan-neural and glial precursors were grown on 3.5-cm tissue culture plates. Cells were treated with purified HTNCre protein as described above. Forty-eight hours after transduction, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells were washed three times with PBS, blocked with 10% normal goat serum in PBS for 20 minutes, and incubated in 5% normal goat serum in PBS overnight with the following primary antibodies: A2B5 (mouse IgM, 1:200; Chemicon, Temecula, CA, http://www.chemicon.com), nestin (clone rat-401, mouse IgG, 1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww), ß-III tubulin (cloneß-III tubulin  goat anti-mouse IgG; FITC goat anti-rabbit IgG; Cy5 goat anti-rat IgG, 1:200; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com; Texas Red-avidin, 1:125; biotin anti-mouse IgM, 1:200; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Mouse primary IgM antibodies were detected using a biotinylated secondary antibody in combination with Texas Red-avidin. For intracellular staining 0.1% Triton was added to the antibody solutions. 4,6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Labeled cells were preserved in Vectashield (Vector Laboratories). Quantitative analysis was carried out by counting the number of immunoreactive cells per total number of viable cells as determined by DAPI staining. Data for each marker were based on at least duplicate cultures with at least 1,000 cells counted.
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RESULTS* Z6 L6 R! `, w
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Conditional Gene Modification by Cre Protein Transduction in Undifferentiated ESCs
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The primary objective of this part of the study was to determine whether cell-permeable Cre recombinase is able to induce site-specific recombination of loxP-modified alleles in ESC-derived neural precursors. To verify the recombination event and determine the efficiency, we decided to use the Z/EG Cre reporter mouse ESC line . HTNCre consists of a basic protein transduction peptide derived from HIV TAT, a nuclear localization sequence, the Cre recombinase protein, and an amino-terminal histidine tag for purification from E. coli. First, we assessed the functionality of the HTNCre protein transduction system in undifferentiated Z/EG ESCs. Incubation of Z/EG ESCs in HTNCre-containing medium resulted in highly efficient eGFP expression in the majority of the cells (Fig. 1B, bottom panel). Flow cytometric analysis revealed that eGFP expression is strictly dependent on the HTNCre concentration (Fig. 1C). Half-maximal activity was achieved with 1 µM, and more than 90% efficiency was obtained after culturing cells in the presence of 2 µM HTNCre. Essentially, all Cre-treated ESCs were devoid of ß-galactosidase activity, as determined by X-Gal staining, whereas untreated control cells were ß-galactosidase-positive (Fig. 1B, right panel). These data indicated that the Z/EG reporter cell line represents a suitable cell line to analyze Cre-mediated recombination using protein transduction.
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Figure 1. Conditional gene modification in Z/EG reporter ESCs induced by cell-permeable Cre recombinase HTNCre. (A): Schematic representation of the Z/EG double reporter construct. The Z/EG ESC line  carries a Cre-dependent double reporter gene. The ubiquitously active CAG promoter drives LacZ expression before and EGFP expression after Cre-mediated recombination. (B): Validation of Cre-mediated recombination by analysis of reporter gene activities in control cells (top panel) and Cre-treated cells (bottom panel). Z/EG ESCs were cultured in medium containing 2 µM recombinant HTNCre for 16 hours. Cells were analyzed 48 hours after protein transduction by fluorescence microscopy and X-Gal staining. (C): Flow cytometric analysis of Cre-treated Z/EG ES. Cells were treated for 16 hours in HTNCre-containing medium as indicated and analyzed 48 hours after transduction. Dot plots show forward scatter versus eGFP fluorescence of control cells and Cre-treated cells using concentrations between 0.25 and 2 µM HTNCre. Histograms depict eGFP fluorescence of untreated cells (black line) and HTNCre-treated cells (green line). Abbreviations: eGFP, enhanced green fluorescent protein; FSC, forward scatter; HTNCre, His-TAT-NLS-Cre; pc, phase contrast; X-Gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.! H( |4 N+ u! f; F
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Neural In Vitro Differentiation of Z/EG Cre Reporter Cells

To analyze Cre protein transduction in mouse ESC-derived neural progeny, Z/EG ESCs were subjected to controlled in vitro differentiation into neural precursors (Fig. 2A). After aggregation to embryoid bodies, spontaneously differentiating neuroepithelial cells were selected in ITSFn medium as described previously . These cells exhibited an elongated bipolar morphology and a predominant glial precursor phenotype. Furthermore, they expressed A2B5, an antigen typically found in murine glial precursors (Fig. 2D). After another 5 days of growth factor withdrawal, the majority of these precursors differentiated into a mixed population of up to 15% oligodendrocytes and 45% astrocytes (Fig. 2E). Immunofluorescence analysis with an antibody to the oligodendrocyte-specific marker O4 revealed cells with characteristic multipolar processes. Anti-GFAP staining showed flat cells with typical astrocytic morphologies (Fig. 2E). These data indicate that Z/EG ESCs have the potential to differentiate in all three neural subpopulations in vitro.; r. _1 r3 e0 x% l3 d
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Figure 2. Neural in vitro differentiation of Z/EG Cre reporter cells. (A): Schematic representation of growth-factor-driven targeted differentiation of Z/EG ESCs in vitro. Z/EG ESCs were differentiated into a pNPC population upon application of FGF-2. Growth factor withdrawal resulted in terminal differentiation preferentially into neurons, whereas application of FGF-2 together with EGF yielded a highly proliferative fraction of gNPCs. These in turn differentiated into a mixed population of astrocytes and oligodendrocytes upon growth factor withdrawal. (B): Image of Z/EG ESC-derived pNPCs exhibiting a typical rosette-like organization of elongated cells. Cells were labeled with an antibody to nestin, an intermediate filament typically expressed in neural stem cells. (C): pNPCs 5 days after growth factor withdrawal, exhibiting typical neuronal morphology with long filamentous processes and immunoreactivity with the ß-III tubulin antibody. (D): Z/EG ESC-derived gNPCs after five passages. Cells exhibited an elongated bipolar morphology and a predominant glial precursor phenotype. These cells expressed A2B5, an antigen typically found in murine glial precursors. (E): After 5 days of growth factor withdrawal, gNPCs differentiated into a mixed population of oligodendrocytes and astrocytes. Immunofluorescence analysis with an antibody to the oligodendrocyte-specific marker O4 (red) reveals cells with characteristic multipolar processes. Anti-GFAP staining (green) shows flat cells with typical astrocytic morphologies. Phase contrast and immunofluorescence images are shown in the left and right panels (B¨CE), respectively. Nuclei are counterstained with DAPI (blue). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; EGF, epidermal growth factor; ES, embryonic stem; FGF-2, fibroblast growth factor-2; GFAP, glial fibrillary acidic protein; gNPC, gliogenic neural precursor cell; pNPC, pan-neural precursor cell.
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Cre Protein Transduction into Neural Precursor Cells Results in Highly Efficient Gene Ablation

Next, we assessed the potential of the HTNCre protein to induce Cre recombination in Z/EG ESC-derived neural cells. First, we analyzed the transduction and recombination potential of HTNCre in proliferating pan-neural and glial precursor cell populations. Z/EG ESCs were differentiated as described above and incubated in medium containing 2 µM HTNCre for 22 hours. Cre-treated cells were analyzed under proliferating conditions (i.e., in the presence of growth factors) by immunofluorescence 48 hours after protein transduction. We observed HTNCre-induced eGFP expression in both pan-neural (Fig. 3A) and glial (Fig. 3B) precursors. HTNCre protein induced recombination in the majority of the cells as determined by eGFP expression. Most of the pan-neural precursor cells turned out to be positive for eGFP and nestin (Fig. 3A). Likewise, immunoreactivity against eGFP and A2B5 was detected in the majority of the Cre-treated glial precursors (Fig. 3B). Southern blot analysis was performed to confirm the recombination event at the DNA level and to obtain a reliable quantification of the HTNCre-induced recombination efficiency. We observed an 8.5-kilobase (kb) band representing the loxP-modified allele and the appearance of a 6-kb band resulting from Cre-mediated recombination (Fig. 3C, 3D). The efficiency of recombination was determined on the basis of the intensities of the respective bands (Fig. 3E). HTNCre induced recombination of the loxP-modified allele in a strictly time- and concentration-dependent manner. Half-maximal efficiency was already obtained after approximately 1 hour in both pan-neural and glial precursors. Recombination efficiency reached a plateau of approximately 95% after 12¨C16 hours of incubation (Fig. 3E). A dose-response analysis was carried out for gliogenic neural precursors using various concentrations of recombinant Cre protein. HTNCre (0.5 µM) induced recombination in approximately 10% of the cells, whereas incubations with 1 and 2 µM resulted in approximately 60% and 95% efficiency, respectively. (Fig. 3F). Notably, the quantitative analysis of eGFP expression did not fully correlate with the quantification based on Southern blot analysis. The percentage of eGFP-positive cells turned out to be reproducibly lower (approximately 50% maximal efficiency) than the percentage of recombined cells as determined by Southern blot analysis. This observation indicates that some of the recombined cells exhibited eGFP expression levels below the threshold of immunofluorescence detection. In conclusion, this part of the study demonstrated that HTNCre is able to efficiently induce site-specific recombination in ESC-derived neural precursors in a time- and concentration-dependent manner. In particular, a 16-hour incubation with 2 µM HTNCre turned out to be sufficient to induce recombination in up to 95% of the neural precursors.: W- n# P( ~7 q7 @

Figure 3. Cre protein transduction into Z/EG ESC-derived neural precursors resulted in highly efficient DNA recombination. (A): Phase contrast and immunofluorescence images of pNPCs after Cre protein transduction using 2 µM His-TAT-NLS-Cre (HTNCre) for 22 hours. Precursor cell morphology and typical rosette architectures were maintained after Cre treatment. eGFP expression in Cre-treated pan neural cell cultures was detected with an anti-GFP antibody (green). The merged image demonstrates the widespread co-expression of nestin (red) and Cre-induced eGFP (green). (B): Phase contrast and immunofluorescence images of Cre-treated ESC-derived gNPCs. Cells were incubated in medium containing 2 µM HTNCre for 22 hours and fixed 48 hours after transduction. Double labeling with antibodies to eGFP (green) and the glial precursor marker A2B5 (red) illustrates successful recombination in the majority of the Cre-treated cells. (C, D): Southern blot analysis of Cre-mediated recombination in pNPCs (C) and gNPCs (D). Cells were treated with 2 µM HTNCre for the indicated periods of time and further cultivated for 48 hours prior to preparation of genomic DNA. An eGFP specific probe was used for the detection of the fl and  allele. (E, F): Quantification of recombination efficiencies by densitometric analysis of Southern blot analysis. (E): Time dependence of HTNCre-induced recombination in pNPCs and gNPCs. This analysis confirms that approximately 90% of the loxP-modified alleles underwent recombination after treatment with 2 µM HTNCre. (F): Concentration and time dependence of HTNCre-induced recombination in gNPCs. Abbreviations: , deleted; fl, loxP-modified; GFP, green fluorescent protein; gNPC, gliogenic neural precursor cell; h, hour(s); pNPC, pan-neural precursor cell; t, time.( ?& G. ~  q  |. ~, l. A- D/ k
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Cre Protein Transduction Does Not Interfere with the Developmental Potential of Neural Precursors* C+ W4 W1 z8 n/ E

Next, we asked whether the treatment with recombinant bacterial Cre preparations might interfere with the developmental properties of ESC-derived precursor cells. For this, we subjected Cre-treated precursor cells to terminal differentiation. Pan-neural precursor cells were incubated with 2 µM HNTCre for 22 hours and cultured for 4 days in the absence of FGF-2 to induce terminal differentiation. Subsequently, cells were analyzed for eGFP fluorescence as well as immunoreactivity with the TUJ1 antibody to the neuronal marker ß-III tubulin. This analysis revealed a high proportion of approximately 40% of eGFP and TUJ1 double-positive cells with typical neuron-like morphology. Only rare cells exhibited TUJ1 immunoreactivity without eGFP expression (Fig. 4A). Along the same line, HTNCre-treated glial precursors were induced to differentiate by a 4-day growth factor withdrawal. Most of the growth factor-withdrawn cells, too, maintained eGFP expression (Fig. 4B). Immunofluorescence analysis using antibodies directed against the oligodendroglial antigen O4 and the astrocyte marker GFAP revealed a mixed population of O4- and GFAP-positive cells, respectively, indicating the bidirectional differentiation capability of Cre-treated glial precursors (Fig. 4C). To confirm that recombined cells were able to generate astrocytes and oligodendrocytes, we analyzed co-expression of Cre-induced eGFP and both glia-specific markers. Indeed, we observed both eGFP-expressing O4-postive cells (Fig. 4D) and eGFP/GFAP double-labeled cells (Fig. 4E). To analyze potentially altered differentiation capabilities quantitatively, we counted differentiated Cre-treated cells and compared these values with those of control cell populations differentiated without Cre treatment. It turned out that Cre-treated pan-neural precursors gave rise to 45 ¡À 5% TUJ1-positive cells, whereas 52 ¡À 5% TUJ1-positive cells were counted after withdrawal of untreated control cells (Fig. 4F). Astrocytic (32 ¡À 5% vs. 45 ¡À 10% in control cells) and oligodendrocyte (12 ¡À 2% vs. 15 ¡À 1%) differentiation were slightly reduced. Overall, we were unable to detect statistically significant changes in lineage-specific differentiation between Cre-treated and untreated control cells. From this observation, we conclude that Cre protein transduction does not interfere with the terminal differentiation of ESC-derived neural precursors into neurons, astrocytes, and oligodendrocytes.9 u* p% P7 A) D

Figure 4. Cre protein transduction did not interfere with the developmental potential of neural precursor cells. (A): The terminal differentiation potential of Cre-transduced pan-neural precursor cells was maintained. Z/EG ESC-derived pNPCs were incubated with 2 µM His-TAT-NLS-Cre (HTNCre) for 22 hours in growth factor-supplemented media. Growth factors were withdrawn 2 days after Cre treatment. Cells were fixed and stained after further cultivation for 3 days. Shown are PC, anti-enhanced green fluorescent protein (anti-eGFP) immunostaining (green), and a merged image demonstrating co-expression of TUJ1 (red) and Cre-induced eGFP (green). (B¨CE): Immunofluorescence analysis of terminal differentiation of Cre-treated Z/EG ESC-derived gNPCs. (B): Glial precursors were incubated with 2 µM HTNCre for 22 hours in growth factor-supplemented media. Growth factors were withdrawn 2 days after Cre treatment. Cells were fixed after further cultivation for 3 days and stained with DAPI (blue) and an eGFP-specific antibody (green). The majority of the cells expressed eGFP after Cre treatment. (C): Expression of GFAP and O4 confirms that Cre-treated glial precursors differentiated into both astrocytes and oligodendrocytes, respectively. (D, E): Double immunofluorescence analyses reveal co-expression of eGFP and O4 (D) and of eGFP and GFAP (E), respectively, indicating successful Cre-mediated recombination in ESC-derived oligodendrocytes and astrocytes. (F): Comparative quantitative analysis of protein marker expression in Cre-treated neural precursor cells (filled columns) versus untreated control cells (open columns) before and after wd. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; gNPC, gliogenic precursor; PC, phase contrast; pNPC, pan-neural precursor; TUJ1, ß-III tubulin; wd, growth factor withdrawal., {" \. I+ f& A' w2 D

Genetic Manipulation of Postmitotic Neuronal Cells by Cre Protein Transduction
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Finally, we assessed whether cell-permeable Cre protein is able to transduce into nondividing ESC-derived postmitotic neurons. For this, we differentiated pan-neural precursors derived from Z/EG reporter ESCs by a 4-day-growth factor withdrawal. Cells were then incubated for 22 hours in media containing 1.5 µM HTNCre protein and analyzed by fluorescence microscopy. We observed a high proportion of ß-III tubulin-positive neurons, which exhibited Cre-induced eGFP expression (Fig. 5A). To rule out the possibility that HTNCre had translocated only into mitotic cells before terminal differentiation, we assessed the mitotic activity of the growth factor-withdrawn cells. For that, we used the incorporation of bromodeoxyuridine (BrdU) to discriminate dividing cells from the postmitotic cells in the differentiated population. Withdrawn Z/EG ESC-derived neuronal cells were cotreated with 1.5 µM HTNCre and 10 µM BrdU. After 22 hours, Cre-containing medium was removed, and 10 µM BrdU was added to the fresh culture medium for an additional 26 hours. On day 7 of differentiation, cells were fixed and stained with antibodies to BrdU, TUJ1, and eGFP (Fig. 5B). We observed only a minor fraction of BrdU-positive cells (25 ¡À 4%), indicating that the majority of the cells were unable to proliferate upon growth factor withdrawal. In particular, we found only occasional TUJ1/BrdU double-positive cells (7.5 ¡À 4.1%), demonstrating that most of the neurons were already postmitotic at the time point of Cre transduction. Overall, we counted 36 ¡À 12% eGFP-positive cells; however, as stated above, this value seems to underestimate the actual recombination efficiency because of the eGFP detection limit. Indeed, Southern blot analysis revealed a fairly higher recombination efficiency of up to 75% (Fig. 5C, 5D). Given the low percentage of BrdU-positive cells, this value indicates that the majority of transduced and recombined neuronal cells were indeed nondividing.) j" M8 J+ I1 y$ V
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Figure 5. Direct delivery of Cre protein into postmitotic neurons efficiently induced recombination. (A): Analysis of ESC-derived neuronal cells 24 hours after Cre protein transduction. Neuronal cells were generated from Z/EG pan-neural precursors (pNPCs) by growth factor withdrawal for 4 days. Differentiated cultures were treated with 1.5 µM HTNCre for 22 hours. Shown are images of PC, as well as immunostaining using antibodies against Cre-induced eGFP (green) and the neuronal marker TUJ1 (red). Untreated cells (top panel) served as controls, demonstrating the specificity of the anti-eGFP immunostaining. (B): Confocal microscopy analysis of Z/EG ESC-derived neuronal cells treated with cell-permeable Cre protein in the presence of BrdU. Neuronal cells were derived from pNPCs by growth factor withdrawal for 4 days. Withdrawn cells were coincubated with 1.5 µM HTNCre and 10 µM BrdU. After 22 hours, Cre-containing medium was removed, and 10 µM BrdU was added to the fresh culture medium for an additional 26 hours. On day 7 of differentiation, cells were fixed and stained with antibodies to BrdU (blue), eGFP (green), and TUJ1 (red). The merged image demonstrates that the majority of the TUJ1-positive/BrdU-negative cells exhibit Cre-induced eGFP expression (arrowheads), indicating that cell-permeable Cre induced recombination in postmitotic neurons. However, Cre-mediated induction of eGFP was also observed in rare BrdU-positive cells (arrow). (C): Quantification of Southern blot analysis (D) of HTNCre-induced recombination in withdrawn pNPCs. pNPCs were cultured for 5 days in the absence of fibroblast growth factor-2 and treated with 1.5 µM HTNCre for the indicated periods of time. Quantification is based on densitometric analysis of Southern blot bands. Abbreviations: BrdU, bromodeoxyuridine; , deleted; fl, loxP-modified; GFP, green fluorescent protein; h, hour(s); HTNCre, His-TAT-NLS-Cre; PC, phase contrast; t, time; TUJ1, ß-III tubulin.4 m3 D$ A) H+ K9 Z* S8 n0 a
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DISCUSSION8 a( {/ q" z; |5 m  ~4 w
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We present a highly efficient, nonviral non-DNA technique for inducing Cre recombinase activity in ESC-derived neural cells by combining two powerful technologies, the Cre/loxP recombination system and protein transduction. We demonstrate that cell-permeable Cre recombinase is able to induce conditional mutagenesis stage-specifically in undifferentiated mouse ESCs, as well as in ESC-derived pan-neural and glial precursor cells. Our method of Cre protein transduction provides good reproducibility and results in exceptionally high recombination efficiencies. Survival and terminal differentiation of the transduced neural cells are not affected by the procedure. Our protocols enable recombination in more than 90% of ESCs and up to 90% of pan-neural precursors and glial precursors, as determined by reporter gene activation and Southern blotting. This stands in contrast to conventional plasmid transfection rates, which, in neural cells, rarely exceed 20% of the surviving cells (data not shown) .: p4 U; ]2 f8 B' b" ?+ G$ n, _, }: w

An important result of our study is that Cre protein transduction is not restricted to dividing stem and precursors cells but may be extended to postmitotic somatic cells. Although viral systems such as Sindbis, Vaccinia, Adeno, and Adeno-associated viruses have been used for the genetic modification of neurons, their application is very laborious and associated with safety concerns. Furthermore, the use of recombinant viral vectors is often limited by cytotoxicity . We here provide proof-of-concept data demonstrating that Cre protein transduction can be used for recombination of the neuronal genome without the necessity of additional genetic modification. Combined with the controlled in vitro differentiation of ESCs, this technology may enable conditional activation or inactivation of any transgene in neuronal networks under stringent temporal control.
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In our paradigm, recombination efficiency is strictly dependent on the duration and concentration of Cre protein incubation. This enables a precise adjustment of the extent of recombination simply by direct titration of the HTNCre protein, desirable, for example, for the analysis of gene functions in a mosaic scenario. In principle, treatment of neural cells with recombinant Cre protein could result in adverse side effects such as toxicity and altered differentiation capabilities. We did not observe obvious toxicity after protein transduction, and Cre-treated pan-neural precursor cells readily differentiated into neuronal cells upon growth factor withdrawal. Likewise, Cre-treated glial precursors were able to differentiate into oligodendrocytes and astrocytic cells. From these observations, we conclude that Cre treatment does not interfere with the developmental potential of the precursor cells.9 M  n7 S. k, i+ ~5 M" i& Y+ ^6 }/ f

Recently, highly efficient delivery of Cre recombinase into neural cells by viral vectors has been reported , can be used to instruct differentiation into a defined neuronal phenotype. The Cre/loxP technology described here may provide a means to efficiently remove such transgenes after successful differentiation without further genetic manipulation, thus enabling the generation of genetically "cleaned" ESC-derived donor cells with a largely unaltered genome.7 M" f2 o( i) G# f

Beyond its applicability in the generation of ESC-derived neural donor cells, Cre protein transduction should be particularly useful for studying the role of individual genes involved in neural development, function, and disease under precise temporal control. Numerous conditional, loxP-modified alleles of genes known to be involved in neural function and development have been reported . Moreover, new candidate genes may be targeted by integration of loxP sites into the ESC genome. The combination of targeted neural differentiation and conditional mutagenesis using the Cre protein transduction system provides a powerful technology to analyze the function of these genes with high fidelity.

DISCLOSURES+ B6 _) U9 u* i" Z

The authors indicate no potential conflicts of interest.. A8 Q" D5 I! H* F! }! N8 B
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ACKNOWLEDGMENTS

We thank Corrinne G. Lobe (University of Toronto) for providing the Z/EG ESC line. In addition, we thank members of the Stem Cell Engineering Group for support and valuable discussions. This work was supported by grants from the Stem Cell Network North Rhine Westphalia (400 004 03), the European Union (LSHB-CT-20003-503005; EUROSTEMCELL), the Volkswagen Foundation (Az I/77864), and the Hertie Foundation. S.H. and F.E. contributed equally to this work.
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