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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

Production of Green Fluorescent Protein Transgenic Embryonic Stem Cells Using the GENSAT Bacterial Artificial Chromosome Library

^aDevelopmental Biology Program and
^bDivision of Neurosurgery, Sloan-Kettering Institute,
^cRockefeller University, New York, New York, USA

Key Words. Neural differentiation • Embryonic stem cell • Bacterial artificial chromosome transgenesis • Fluorescent reporter

Correspondence: Mark J. Tomishima, Ph.D., Developmental Biology & Neurosurgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 256, New York, New York 10021, USA. Telephone: 212-639-3913; Fax: 646-422-2062; e-mail: tomishim{at}mskcc.org

Received on March 23, 2006; accepted for publication on September 14, 2006.

First published online in STEM CELLS EXPRESS  September 21, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated. Specific GFP expression in transgenic cell lines was confirmed during neural differentiation marking neural stem cells, neuronal precursors, and glial progeny by Hes5, Dll1, and GFAP, respectively. GFP was dynamically regulated in ES cell progeny in response to soluble factors that inhibit Notch signaling and a factor that directs astroglial fate choice. Our protocols provide a simple and efficient strategy to utilize the whole GENSAT BAC library to create hundreds of novel fluorescent cell lines for use in ES cell biology.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The creation of transgenic embryonic stem (ES) cells that express green fluorescent protein (GFP) under the control of specific promoters has emerged as a powerful method for monitoring gene expression in live stem cells or their differentiated progeny. Traditionally, transgenic stem cells were created by the site-specific targeting of GFP into a gene of interest or by randomly integrating DNA containing a specific promoter or enhancer elements adjacent to GFP [1]. There are strengths and weaknesses to each strategy. Targeted insertion usually results in correct transgene expression but is labor-intensive and results in a cell line with only one copy of the gene of interest, since GFP replaces the other endogenous copy. Random integration of promoter-driven GFP constructs requires mapping the promoter structure. After mapping, recombinant DNA techniques must be used to create truncated promoters that typically are smaller than 20 kilobases (kb) due to the practical limitations of conventional cloning methods [2]. These smaller, randomly integrated transgenes are frequently dysregulated [3–7].

One strategy to bypass these limitations is the use of bacterial artificial chromosome (BAC) transgenes. BACs are composed of large (up to approximately 350 kb) pieces of genomic DNA. In mice and zebrafish, BAC-based reporters properly regulate gene expression irrespective of integration site when introduced randomly into the genome as a transgene [3–7]. Therefore, BAC transgenesis combines the advantages of "knock-in" strategies using homologous recombination (specificity of expression) with that of transgenic approaches (multiple copies and ease of use). Moreover, unlike knock-in strategies that modify or eliminate the coding sequence, the genomic locus being studied is not modified. The increased transgene fidelity observed with BACs is likely due to the BAC's large size, which insulates the integrated transgene from the effects of the surrounding chromatin structure. The larger size of BACs might also provide a more complete set of regulatory sequences [3–7]. However, while BACs have been used extensively for creating transgenic mice, little work has been done to develop BAC transgenesis for use in ES cells and in vitro differentiation assays.

Here, we show that BACs can be readily introduced into ES cells to produce cells that express GFP at defined stages of neural differentiation. Our work provides the tools sufficient to retrofit the entire GENSAT library, allowing stem cell researchers rapid access to more than 400 GFP-expressing BACs. In the postgenomic era, ES-cell-based GFP expression libraries should greatly facilitate efforts aimed at the understanding of gene function and could provide many cell lines useful in high throughput screens for developmental processes or drug discovery.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
GENSAT BACs
The GENSAT BAC for GFAP::GFP was constructed from RP24-331K9, Hes5::GFP from RP24-341I10, and Dll1::GFP from RP23-306J23 (see http://www.gensat.org for more information on each BAC). GENSAT BACs can be obtained from ATCC (http://www.atcc.org).

Retrofitting BACs for Mammalian Selection
The retrofitting protocol was adapted from Liu et al. [8] (see Fig. 1 for flowchart). Comprehensive protocols can be found in the supplemental materials online. Briefly, GENSAT BACs were moved from Escherichia coli strain DH10B (Fig. 1A) to the E. coli strain EL350 (Fig. 1B), a strain that inducibly expresses the Cre recombinase [3]. A selection cassette flanked by loxP sites was excised from the plasmid pL452 and gel-purified. This linear cassette was electroporated into Cre-expressing EL350 (Fig. 1C), and selection was performed on LB plates containing chloramphenicol and kanamycin (12.5 µg/ml each). Southern blots were performed to verify that the selection cassette had incorporated into the loxP site in the BAC backbone. During the course of these experiments, we discovered that low-level Cre expression in EL350 led to a small population of BACs that had excised the selection cassette despite continued kanamycin selection (Fig. 1). Therefore, we transferred the modified BACs from EL350 back to DH10B (Fig. 1E) before preparing large-scale DNA preparations for ES cell electroporation.

View larger version (42K): [in this window] [in a new window]   Figure 1. Schematic representation of the bacterial artificial chromosome (BAC) retrofitting protocol. GENSAT BAC DNA is isolated from the bacterial strain DH10B (A) and is transformed into electrocompetent EL350 (B). Electrocompetent EL350 that express Cre and contain the BAC of interest are electroporated with a floxed kan/G418 resistance cassette. Electroporated bacteria are plated on kanamycin- and chloramphenicol-containing plates to select for bacteria containing modified BACS. To verify that the kan-resistant bacteria contained the kan cassette in the loxP site present in the BAC backbone, we re-expressed Cre recombinase and verified that the bacteria were sensitive to kanamycin (D). Southern blots showed that a fraction of the BACS present in EL350 had excised the cassette (blue arrow in [C]), presumably due to leaky expression of the Cre recombinase in the bacteria. Therefore, we isolated DNA from EL350 and transformed the modified BAC into DH10B (E). This eliminated the appearance of the excised kan cassette (note the lack of the blue band in lane E). The green arrow on the Southern blot shows a background band present in all lanes (including the parental BAC in DH10B), whereas the red arrow shows that the kanamycin cassette is targeted to an EcoRI fragment of the predicted size (approximately 12 kilobases). Abbreviation: GFP, green fluorescent protein.

ES Cell Propagation
We used low-passage (between 10 and 30) CJ7, R1, and E14 ES cells in this study and maintained them primarily on Neo-resistant MEFs (Specialty Media). Transgenic ES cells were maintained in 500 µg/ml G418 on Neo-resistant MEFs. Prior to differentiation, BAC transgenic ES cells were passaged onto gelatin-coated tissue culture dishes for 1 day to remove MEFs, and selection was removed during neural induction.

Neural Induction
ES cells were directed to neural cell fates using two different protocols [10, 11]. For the GFAP::GFP specificity experiments, we used a modified serum-free floating culture of embryoid-body-like aggregates (SFEB) protocol [11] to cause neural induction, then triturated and plated single cells in N2 medium with or without 50 µg/ml ciliary neurotrophic factor (CNTF) in tissue culture dishes for 14 days. The medium used for the SFEB protocol was a 1:1 mixture of SRM and N2 (components listed in [10,12]). The medium was changed at day 3, and spheres were used at day 6. The SFEB protocol was also used to test Dll1::GFP and Hes5::GFP transgene sensitivity to the Notch inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl]-S-phenylglycine tert-butyl ester (DAPT) (10 µM; EMD Biosciences, Darmstadt, Germany, http://www.emdbiosciences.com, catalog number 565770). The MS5 stromal feeder protocol [10] was used to count the number of cells coexpressing markers for Dll1::GFP and Hes5::GFP and to test the responses of 24 independent Dll1::GFP and 24 Hes5::GFP clones (Fig. 2B) to DAPT. All data are derived from at least three independent experiments unless noted.

View larger version (41K): [in this window] [in a new window]   Figure 2. Polyclonal GENSAT bacterial artificial chromosome (BAC) transgenic ES cells regulate GFP expression in response to soluble factors known to regulate the endogenous gene. (A): Neural progeny from Dll1::GFP and Hes5::GFP polyclonal ES cells are differentially regulated by the Notch inhibitor DAPT. Neural spheres exhibited increased fluorescence (Dll1::GFP) or decreased fluorescence (Hes5::GFP) after exposure to DAPT. Spheres were dissociated, and the number of GFP-positive cells was quantitated by flow cytometry. Scale bar = 200 µm. (B): Twenty-four independently derived Dll1::GFP and Hes5::GFP monoclonal cell lines were derived and differentiated into neural cells before exposure to DAPT. In every cell line that expressed GFP, the fluorescence increased for Dll1::GFP and decreased for Hes5::GFP (C): Neural cells derived from GFAP::GFP polyclonal ES cells fluoresce after extended culture, and there is an increased proportion of GFP-positive cells in cultures supplemented with CNTF. Scale bar = 500 µm. Abbreviations: CNTF, ciliary neurotrophic factor; DAPI, 4',6-diamidino-2-phenylindole; DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl]-S-phenylglycine tert-butyl ester; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.

Antibodies for Immunofluorescence
The rabbit polyclonal antibody against GFP was purchased from Molecular Probes (Eugene, OR, http://probes.invitrogen.com) (A11122 [GenBank] , 1:600). A monoclonal antibody against glial fibrillary acidic protein (GFAP) was purchased from Chemicon (Temecula, CA, http://www.chemicon.com) (MAB 3580 1:1,000). The TuJ1 antibody was purchased from Covance (Princeton, NJ, http://www.covance.com) (MMS-435P). Goat anti-mouse Alexa Fluor 568 (A11004 [GenBank] ) and goat anti-rabbit Alexa Fluor 488 (A11008 [GenBank] ) secondary antibodies were purchased from Molecular Probes (1:500).

Fluorescence-Activated Cell Sorting for Quantification
For the quantification experiments in Figures 1A, 1B, and 3C, Dll1::GFP and Hes5::GFP SFEBs were expanded for 3 days before the addition of 10 µM DAPT to half of the samples from days 3 to 6. On day 6, control and DAPT-exposed neural spheres were dissociated in 0.05% trypsin-EDTA for 7 minutes before fluorescence-activated cell sorting (FACS) into GFP-positive and -negative populations on a MoFlo Sorter (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com).

View larger version (57K): [in this window] [in a new window]   Figure 3. Analysis of clonally derived Notch pathway transgenic lines Dll1::GFP clone 10 and Hes5::GFP clone 1. (A): Dll1::GFP clone 10 expresses GFP primarily in TuJ1-positive cells and not Nestin-positive cells. (B): Hes5::GFP clone 1 expresses GFP primarily in Nestin-positive cells and not TuJ1-positive cells. Scale bar = 50 µm. (C): Neural spheres of Dll1::GFP clone 10 and Hes5::GFP clone 1 were exposed to the Notch inhibitor DAPT after neural induction, and the number of GFP-positive cells within the spheres was quantitated by flow cytometry. (D): Flow cytometry was used to separate GFP-negative and -positive cells followed by quantitative polymerase chain reaction for Dll1 and Hes5. The data shown are the fold increase in the amount of Dll1 or Hes5 mRNA in the GFP-positive fraction relative to the GFP-negative fraction after normalization (see Materials and Methods). Abbreviations: DAPT, N-[N-(3,5-difluoro-phenacetyl-L-alanyl]-S-phenylglycine tert-butyl ester; GFP, green fluorescent protein.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Integration Site Effects on GENSAT BAC Transgene Expression
In mice, BACs have been shown to direct specific transgene expression independent of the BAC's integration site [3–7]. However, transgenic mice are created by injection of BAC DNA directly into fertilized mouse eggs. Here, we modified GENSAT BACs by introducing a selection cassette followed by electroporation of the modified BAC into ES cells, drug selection, and isolation of transgenic stem cells. To examine possible effects of the integration site on transgene expression in ES cells, we electroporated ES cells with three separate BACs and passaged all drug-resistant ES cells as polyclonal cell lines. In this way, we could assay hundreds of BAC transgene integration sites by differentiating these pooled, polyclonal cell lines derived from each BAC.

We first examined the expression of two Notch-related genes after neural induction. It is thought that Notch ligands are expressed on the surface of neuroblasts where they interact with the Notch receptors to regulate the pool of neural stem cells [14–17]. To watch Notch signaling in differentiating neural cells, we created transgenic ES cells with delta-like1::GFP (Dll1—one of the Notch ligands) and Hes5::GFP (activated by Notch signaling). Neural induction of transgenic Dll1::GFP and Hes5::GFP ES cells using a serum-free culture system [11] resulted in spherical aggregates with unique patterns of GFP expression within spheres (Figs. 2A, 4). In Hes5::GFP spheres, the foci of GFP expression were usually located on the outside of neural spheres and in regions where two spheres contacted each other. Dll1::GFP progeny showed scattered expression of GFP throughout the sphere (Figs. 2A, 4). Inhibiting Notch signaling should reduce downstream Notch gene expression (Hes5) and increase Notch ligand expression (Dll1). Notch inhibition should cause Hes5-expressing neural stem cells to commit to a neuronal fate, activating Dll1 expression [15–17]. Therefore, we predicted that the Notch inhibitor DAPT should reduce GFP expression in Hes5::GFP spheres and enhance GFP expression in Dll1::GFP spheres if the BAC transgenes were correctly regulated. To test this prediction, we differentiated Dll1::GFP and Hes5::GFP ES cells into neural spheres. From days 3 to 6, half of each culture was exposed to DAPT. Direct microscopic observation revealed increased fluorescence in Dll1::GFP spheres that received DAPT when compared with control cultures without DAPT (Fig. 2A). Conversely, Hes5::GFP spheres exposed to DAPT showed decreased fluorescence (Fig. 2A). To quantitate this observation, neural spheres were dissociated on day 6, and FACS analysis was performed. The number of GFP+ cells derived from Dll1::GFP spheres in control cultures was 5.5 ± 2.1% and increased to 9.9 ± 2.0% in cultures exposed to DAPT. In Hes5::GFP control spheres, 5.0 ± 2.0% expressed GFP compared with 0.3 ± 0.2% when exposed to DAPT.

View larger version (15K): [in this window] [in a new window]   Figure 4. GFP expression patterns in Hes5::GFP clone 1 and Dll1::GFP clone 10 neural spheres. Neural spheres were cultured for 7 days in vitro before live imaging of GFP fluorescence. Five representative spheres of each cell line are shown. Scale bar = 400 µm. Abbreviation: GFP, green fluorescent protein.

In contrast to the Notch-related transgenic cell lines, the GFAP::GFP polyclonal cell lines did not fluoresce after neural induction. GFAP::GFP should be expressed in astrocytes, a neural cell type that appears later in development and after extended culture during in vitro differentiation of ES-derived or primary neural precursors. Astrocyte production can be further enhanced by exposure to the growth factor CNTF [12]. In agreement with these predictions, extended culture of GFAP::GFP ES-derived neural precursors led to the appearance of cells that were immunoreactive for both GFP and GFAP, and the addition of CNTF further increased the number of double-positive cells (Fig. 2C). Given the polyclonal nature of the ES cells used in these experiments, our data indicate that BAC transgenes are regulated correctly in ES cells without obvious influence from the BAC integration site.

Specificity of Clonal BAC Transgenic Cell Lines
To further assess ES cell transgene specificity, we isolated and characterized single-cell-derived BAC transgenic ES cell clones. The percentage of drug-resistant clones that expressed GFP after neural induction varied depending on the BAC selected but was at least 10% (for example, see Fig. 2B, both Dll1::GFP and Hes5::GFP yielded 10 fluorescent colonies out of 24 total colonies, or approximately 40%). While GFP expression patterns appeared to be identical among multiple lines for each BAC examined, there were clear differences in the strength of GFP expression noted between lines. For each BAC, one representative cell line was studied in detail and is presented below.

Dll1::GFP Clone 10
Similar to the results obtained with mixed clones, Dll1::GFP clone 10 showed widespread scattered expression within differentiating neural colonies (Fig. 4). Immunofluorescent analysis of attached cells (Fig. 3A) demonstrated that 87.6 ± 2.3% of the GFP-positive cells were immunopositive for TuJ1, while only a minority of the cells expressed GFP only (9.7 ± 1.9%) or TuJ1 alone (2.7 ± 0.5%). Few of the GFP-positive cells coexpressed Nestin (7.5 ± 1.2%%+ - Fig. 3). Time-lapse microscopy revealed that Dll1::GFP-positive cells were motile and divided frequently during neural differentiation (supplemental online Video 1). Immunofluorescence analysis with antibodies against proliferating cell nuclear antigen and GFP confirmed our observation that many of the Dll1::GFP-positive cells were dividing (data not shown). This suggests that Dll1 is expressed in neuroblasts, dividing precursor cells that adopt postmitotic neuronal fates. Upon further differentiation we observed GFP-positive cells with mature neuronal morphologies (supplemental online Video 2).

To verify that GFP expression increases when inhibiting Notch, Dll1::GFP clone 10 neural spheres were exposed to DAPT as outlined above. Fluorescence microscopy revealed increased fluorescence after exposure to DAPT, and flow cytometry showed that the number of GFP-positive cells more than doubled to 45 ± 7.5% in cultures with DAPT when compared with control cultures (22.1 ± 3.7%, Fig. 3C). We also found that GFP expression identified cells expressing high levels of Dll1 transcripts. GFP-positive and -negative cells were separated by flow cytometry before performing quantitative PCR for Dll1 (Fig. 4D). We observed a 20.6-fold increase in Dll1 transcripts in Dll1::GFP-positive cells relative to Dll1::GFP-negative cells (see Materials and Methods).

Hes5::GFP Clone 1
Most Hes5::GFP-positive cells had a more flattened appearance and coexpressed Nestin (81.0 ± 2.5%, Fig. 3B). Fewer cells expressed Nestin only (16.1 ± 3.2%) or GFP only (2.9 ± 1.2%). Hes5::GFP clone 1 cells were mostly negative for the neuronal marker TuJ1 (11.2 ± 1.7%, Fig. 3B). Hes5::GFP neural spheres primarily had fluorescent cells clustered on the outside of spheres (Fig. 4), and incubation of Hes5::GFP neural spheres with DAPT decreased the proportion of GFP-positive cells from 33.0 ± 8.3% to 4.62 ± 2.2% (Fig. 3C). Separation of Hes5::GFP-positive and -negative cells by FACS followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that Hes5 transcripts were highly enriched in the GFP-positive fraction (Fig. 3D). Quantitative PCR experiments showed that Hes5::GFP-positive cells contained 19.7-fold more Hes5 mRNA relative to the GFP-negative population (see Materials and Methods).

GFAP::GFP Clone 10
The ES cell line GFAP::GFP clone 10 only fluoresced after culture conditions that caused the appearance of astrocytes, similar to the GFAP::GFP mixed clones described above. Indirect immunofluorescent analysis using antibodies against GFP and GFAP (Fig. 5) demonstrated a striking degree of colocalization (89.5 ± 4.3%), although cells expressing only GFP (5.5 ± 1.6%) or GFAP (4.7 ± 2.9%) appeared in the cultures. These single-labeled cells were nearly always found near each other and the double-positive cells.

View larger version (39K): [in this window] [in a new window]   Figure 5. GFAP::GFP clone 10 expressed GFP in GFAP immunoreactive cells. Immunofluorescence demonstrated that most GFP-positive cells also express GFAP. The merged images include 4',6-diamidino-2-phenylindole to demonstrate the specificity of the GFP and GFAP staining. The high magnification images in the lower portion of the panel show the boxed regions in the top portion of the panel. Scale bar = 200 µm (top) or 50 µm (bottom). Abbreviations: GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

Multiple lines of evidence suggest that the BAC transgenic ES cell lines function correctly. (a) Timing of expression: we did not find any ES cell clones that expressed appreciable amounts of GFP as ES cells (data not shown). As both Notch BAC ES cell lines differentiated into neural cells, they began to express GFP within the first week of differentiation when Notch components are detectable by RT-PCR (data not shown). However, the GFAP::GFP lines only began to express GFP into the third week of differentiation, when astrocytes begin to develop in the cultures [10]. (b) Patterns of GFP expression: each BAC has a characteristic pattern of GFP expression in differentiating ES-derived neural colonies. Hes5::GFP is expressed in continuous patches that are largely on the outside of neural spheres, whereas Dll1::GFP is scattered throughout the spheres (Fig. 4). GFAP::GFP is not expressed in neural spheres that contain neural progenitors and neurons but not glial cells (data not shown). As noted above, GFP-positive astrocytes (GFAP+) only appeared after extended culture. (c) Response to soluble factors: the Notch-related Hes5::GFP and Dll1::GFP neural spheres displayed opposing phenotypes after exposure to DAPT, a chemical inhibitor of the Notch pathway. DAPT exposure reduced Hes5::GFP expression while it increased Dll1::GFP expression as predicted. In GFAP::GFP lines, the addition of a growth factor that instructs astrocytic cell fate (GFAP+ cells) increased the number of GFP-positive astrocytes compared with control cultures that were not exposed to CNTF. (d) GFP expression in different cell types: immunofluorescent experiments (Figs. 3, 5) demonstrated that Hes5::GFP and Dll1::GFP labeled different neural cell populations. Dll1::GFP showed a high degree of colocalization with TuJ1, consistent with neuronal labeling (Fig. 3A). Time-lapse microscopy revealed that many of these cells were actively dividing, consistent with the hypothesis that the Dll1::GFP-positive cells are mainly composed of neuroblasts and postmitotic neurons (supplemental Videos 1 and 2). Hes5::GFP, on the other hand, primarily labeled Nestin+ cells (Fig. 3B), and GFAP::GFP-positive cells were around 95% GFAP-positive astrocytes. (e) mRNA enrichment in GFP+ cells: Dll1::GFP and Hes5::GFP-positive neural cells contained approximately 20-fold higher levels of Dll1 and Hes5 mRNA compared with GFP-negative cells from Dll1::GFP and Hes5::GFP cell lines, respectively (Fig. 3D). Taken together, our data demonstrate that GENSAT BACs can be used to rapidly produce ES cell lines that fluoresce during the transcription of desired genes.

BAC transgenes have been shown to more reliably report on an endogenous gene in vivo when compared directly with smaller transgenes [3–7]. These studies showed that BAC transgenes are less susceptible to mosaic or position effect variegation, which is likely due to transgene silencing. It is also possible that the more extensive amount of sequence present in BACs is required for robust transgene expression, since even the inclusion of insulator elements on conventional transgenes only partially rescues mosaic expression [4]. We examined the effects of integration site in two ways: by producing polyclonal transgenic ES cell populations that consisted of all G418-resistant colonies passaged together, and by assaying 48 independently-derived clones for transgene activity (Fig. 2). In each case, we observed little influence of the integration site on transgene fidelity. For example, we did not observe GFP expression in the ES cell stage of any BAC examined, nor did we observe inappropriate GFAP::GFP expression after neural induction. While expression fidelity did not appear to be influenced by integration site, GFP expression levels did vary between clones. For the GFAP::GFP clone 10, we observed astrocytes (GFAP+) that did not express GFP (Fig. 5) and vice versa. There are a few possible reasons for this observation. One possibility is that the transgene is being silenced in a minority of the cells. Another possibility is that there are differences between promoter activity and protein stability. It is important to remember that all conventional transgenes express GFP during transcription of the gene of interest. Post-transcriptional regulation mechanisms, such as mRNA and protein stability, are not accounted for in such transgene strategies. Therefore, we believe that this is the likely explanation for the observation of single GFP- and GFAP-stained cells. Further experiments will be necessary to clarify this discrepancy.

The routine production of ES reporter cell lines will be crucial for many applications, even if some transgene silencing does occur. Such cell lines will allow the enrichment of specific neural cell types and developmental stages for preclinical applications in animal models of disease. BAC transgenic ES cells will also permit the identification and quantification of specific ES-derived neurons in response to extracellular stimuli that damage or protect particular neuron types. Such neuron types could include midbrain dopamine neurons or spinal motor neurons that can be readily obtained from mouse ES cells [20–22] and that are critical for the study of Parkinson disease and amyotrophic lateral sclerosis. Additional important applications will include genomic or proteomic studies of FACS-purified cell types within mixed neural cultures, the prospective isolation of ES-derived neural progeny for cell fate analysis and transplantation, and large-scale chemical, RNA interference, or cDNA screens to define molecules involved in stem cell fate specification.

The GENSAT BAC library was initially constructed to identify cell types during nervous system development [18, 19]. This project has provided hundreds of BAC transgenic mice that label defined classes of neural cells (http://www.gensat.org and [18, 19]). Our data show that this existing resource can be readily modified to study neural differentiation of ES cells in vitro. One advantage of using this existing BAC library is that the GFP transcriptional fusions have already been engineered, constructed, and tested in the production of transgenic mice that are available for distribution. The only modification necessary is to retrofit each BAC with a mammalian selection cassette. The efficiency of the technique presented here suggests that it should be possible to generate BAC transgenic ES cell lines for the whole GENSAT library. Access to GFP-expressing ES cell lines for more than 400 central nervous system related genes will provide an essential resource for the stem cell field.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
We thank Nathaniel Heintz (Rockefeller University) for conversations, advice, and reagents to perform these experiments. In addition, we thank Neal Copeland and Nancy Jenkins (NCI) for providing the reagents necessary to retrofit the BACs. The entire Studer Lab provided helpful feedback, but Yechiel Elkabetz and Sabrina Desbordes deserve special thanks for constructive discussions. We also thank Jan Hendrikx and Cris Bare for help with flow cytometry and Agnes Viale and Juan Li for help with qPCR. Dennis Kunkel Microscopy, Inc. (Kailua, HI, http://www.denniskunkel.com) kindly provided the E. Coli image used in Figure 1.

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