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作者：Mark J. Tomishimaa,b, Anna-Katerina Hadjantonakisa, Shiaoching Gongc, Lorenz Studera,b 8 [- L: _! a2 l" F; U) L: b- g0 b
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【摘要】8 ^) ?: F& W0 C. _7 z3 x1 |
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 ) 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.
( J+ s" L" q# o4 h6 |: b- G 【关键词】 Neural differentiation Embryonic stem cell Bacterial artificial chromosome transgenesis Fluorescent reporter
4 B H& J4 N9 @) V# D J INTRODUCTION
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9 B2 n, N- e$ ^0 a1 RThe 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 .
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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 . 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.; Q4 b9 b1 H+ E: e( @
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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.
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MATERIALS AND METHODS3 p w9 w0 V" {$ J ?2 o6 [7 u% E8 s0 l
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GENSAT BACs
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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).
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Retrofitting BACs for Mammalian Selection# q6 ^: s( i Z ^( r# Y
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The retrofitting protocol was adapted from Liu et al. . 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.& e3 |7 m, `9 K
* Q+ L6 h0 O$ i" [6 T6 y3 qFigure 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 ), 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.6 D6 b% n8 v* N8 L; Z2 E2 |
K4 C9 Z0 d! E4 } `Preparing Retrofitted BAC DNA to Electroporate ES Cells/ x8 E7 s$ r) @1 K" J4 n
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To produce purified DNA to electroporate into ES cells, we used the Psi Clone Big BAC DNA kit (Princeton Separations, Adelphia, PA, http://www.prinsep.com, PP-121) and followed the manufacturer's protocol. Approximately 100 µg of BAC DNA was electroporated into 5.6 x 106 CJ7 or E14 ES cells. Electroporation was performed as described with a few modifications. Briefly, 7 x 106 ES cells were resuspended in 1 ml of room-temperature phosphate-buffered saline (PBS), and 800 µl of this cell suspension was added to the DNA preparation. Electroporation was performed in a 4-mm cuvette at 250 V, 500 µF, and cells were left at room temperature for 10¨C20 minutes before plating onto two 10-cm dishes of Neo-resistant mouse embryonic fibroblasts (MEFs) (Primary Mouse Embryo Fibroblasts, PMEF-N; Chemicon/Specialty Media, Phillipsburg, NJ, http://www.specialtymedia.com). Selection in 500 µg/ml G418 was started 2 days after electroporation, and colonies were manually picked and grown clonally after 7¨C10 days of selection. For experiments using polyclonal pools of ES cells, all of the colonies from an electroporation were passaged together as a single line. Each polyclonal line was derived from the following number of clones: Dll1::GFP, 115; GFAP::GFP, 123; and Hes5::GFP, 281.' h. G+ ]4 {, R7 R+ I1 \! q
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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.
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Neural Induction
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ES cells were directed to neural cell fates using two different protocols 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./ i' N4 E; ?# Q+ b; E" V$ i
' a0 J$ g+ J: T/ y; ]4 `- h8 }$ s- ?) XFigure 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--S-phenylglycine tert-butyl ester; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.) L: n/ w3 V3 A2 l% w% O/ l$ w
' C: \, y8 v4 B4 K0 J7 ?6 uIndirect Immunofluorescence
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# i4 R) I' K0 d& k2 K3 S6 wCultured cells were washed once with PBS before fixation with 4% paraformaldehyde/0.15% picric acid for 15 minutes at room temperature. Cells were washed three times with PBS before permeabilization with wash buffer for at least 2 minutes (0.3% triton X-100, 1.0% bovine serum albumin in PBS). Primary antibody (diluted in wash buffer) was added to the cells for 3 hours at room temperature. Cells were washed three times with wash buffer before the addition of secondary antibody (diluted in wash buffer) for 1 hour. Cells were washed three times with wash buffer and once with PBS and were stored at 4¡ãC in 50% PBS and 50% glycerol before acquiring images.
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Antibodies for Immunofluorescence
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i$ A' M* O+ R: x: S4 [The rabbit polyclonal antibody against GFP was purchased from Molecular Probes (Eugene, OR, http://probes.invitrogen.com) (A11122 ) secondary antibodies were purchased from Molecular Probes (1:500).* u* [' ~+ o+ U. z" x
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Fluorescence-Activate
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发表于 2009-3-5 01:01
发表于 2009-6-16 13:04
