Short Technical Reports
Trophoblast-specific gene manipulation using lentivirus-based vectors Pantelis Georgiades1,2, Brian Cox2,3, Marina Gertsenstein4, Kallayanee Chawengsaksophak2, and Janet Rossant2,3 1University
of Cyprus, Nicosia, Cyprus, 2Hospital for Sick Children, Toronto, of Toronto, Toronto, and 4Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
3University
BioTechniques 42:317-325 (March 2007) doi 10.2144/000112341
The trophoblast layers of the mammalian placenta carry out many complex functions required to pattern the developing embryo and maintain its growth and survival in the uterine environment. Genetic disruption of many gene pathways can result in embryonic lethality because of placental failure, potentially confusing the interpretation of mouse knockout phenotypes. Development of tools to specifically and efficiently manipulate gene expression in the trophoblast lineage would greatly aid understanding of the relative roles of different genetic pathways in the trophoblast versus embryonic lineages. We show that short-term lentivirusmediated infection of mouse blastocysts can lead to rapid expression of a green fluorescent protein (GFP) transgene specifically in the outer trophoblast progenitors and their later placental derivatives. Efficient trophoblast-specific gene knockdown can also be produced by lentivirus-mediated pol III-driven short hairpin RNA (shRNA) and efficient trophoblastspecific gene knockout by pol II-driven Cre recombinase lentiviral vectors. This lentivirus lineage-specific infection system thus facilitates both gain and loss of function studies during placental development in the mouse and potentially other mammalian species.
INTRODUCTION During early mammalian development, the first lineage distinction occurs at the blastocyst stage, with the separation of the outer trophectoderm (TE) from the inner cell mass (ICM) (1). The TE goes on to form the extraembryonic ectoderm and ectoplacental cone after implantation; these trophoblast tissues form the majority of the fetal-derived components of the mature placenta. The ICM gives rise to the fetus itself, as well as some other extraembryonic cell types. The trophoblast supports the growth and development of the fetus in the uterine environment, by facilitating nutrient, gas, and waste exchange in the placenta, ensuring immune protection of the fetus, and producing a variety of hormones and cytokines that influence both maternal and fetal systems (2). In addition, the early postimplantation trophoblast, prior to mature placental formation, is a key player in the complex signaling interactions required to establish the basic body Vol. 42 ı No. 3 ı 2007
axes in the developing embryo itself (3). Given its complex roles, it is not surprising that many genetic pathways impinge on trophoblast development and function. However, many of these pathways are also involved in the development of the embryo itself; genetic knockout studies often fail to separate the different roles (4). Indeed, in some well-known cases, genes have been incorrectly proposed to be necessary for the development of specific embryonic tissues, based on phenotypes that later turned out to be entirely secondary to defects in the placenta (5,6). Embryonic stem (ES) cell-derived embryos, made by aggregating ES cells with tetraploid embryos, can be used to separate the action of mutations in embryonic versus extraembryonic lineages (7). However, this system fails to separate functions in the trophoblast versus the extraembryonic endoderm and does not address very early lineage specific effects. Trophoblast stem (TS) cell chimeras could in theory be used to manipulate gene expression specifi-
cally in the trophoblast, but it is difficult to obtain consistently high levels of chimerism within the trophoblast to make this a viable option (8). Gain of function or rescue of gene function analysis could be achieved by standard transgenesis, using trophoblast-specific promoter-driven transgenes. Loss of function could be achieved using the same promoters to drive Cre or Flp recombinases for conditional gene targeting (9). However, there are limited numbers of well-defined trophoblastspecific promoters and none that is expressed throughout the trophoblast lineage at all stages of development. These approaches are also tedious and slow, requiring multiple rounds of mouse breeding. We aimed to develop a simple technique for rapid and facile gene function analysis in the trophoblast, to complement the use of ES-cell derived mice. While non-lentiviral retroviruses have long been known to infect embryos at the preimplantation stage (10), lentivirus vectors have been shown to infect early mouse embryos more efficiently; up to 80% of embryos were reported to show widespread transgene expression at later stages after infection of one-cell, two-cell, or morulae (11). The same vectors can infect ES cells and seem to be less prone to silencing than other retrovirus vectors (12). We reasoned that infecting not morulae but blastocysts could prove an effective way of achieving trophoblast-restricted gene expression from the blastocyst stage onwards. Passage of large molecules into the ICM and the blastocoelic cavity is known to be prevented by the epithelial tight junctions of the TE. This suggested that viral particles would also be excluded, thus allowing infection of only the outside TE cells. There has been one report that describes the use of a lentiviral vector to infect the TE of rhesus monkey (Macaca mulatta) at the blastocyst stage (13). The blastocysts were infected by microinjection into the blastocoel, which led to the infection of the both the TE and the primitive endoderm derived tissues. We present here a methodology for the infection of only the TE layer by culturing the blastocysts in media with lentiviral vectors. www.biotechniques.com ı BioTechniques ı 317
Short Technical Reports MATERIALS AND METHODS Generation of Virus Plasmids for generation of the FUGW UbiC enhanced green fluorescent protein (EGFP) (11) and FG12 U6 RNA interference (RNAi) LacZ lentivirus (14) were kindly provided by Drs. Carlos Lois (Division of Biology, California Institute of Technology, Pasadena, CA) and Xiao-Feng Qin (University of Texas M.D. Anderson Cancer Center, Houston, TX), respectively. The lentivirus expressing Cre was generated by cloning the nuclear localized improved Cre recombinase (iCre) gene (15) into the EF.v.CMV. GFP vector (16) (accession no. JHU55; ATCC, Manassas, VA, USA) at the EcoRV restriction site. Virus was grown as published (17), and viral titer was determined as published (18). Animals and Preimplantation Stage Embryo Manipulation Mouse embryos were collected at either E2.5 or E3.5 from timed mated mice by flushing the oviduct or uterine horns with Specialty Media® M2 medium (Millipore, Billerica, MA, USA) as described (19). ICR outbred mice were mated to generate blastocysts for infection with the FUGW UbiC EGFP virus. Male mice
Table 1. Zona Presence and Viral Exposure Time Effect on Rate of Infection of Cultured Blastocysts Virus
Zona
Exposure Time (h)
Embryos (No.)
GFP Positive
TE Only
FUGW
Yes
4
3
0
0
FUGW
No
4
7
7
7
FUGW
No
5
8
8
8
FG12
No
6
18
18
18
FUGW
No
24
14
14
14
Effect of zona presence and viral exposure time on the rate of infection of cultured blastocysts was imaged 24 h from the start of exposure time with FUGW. GFP, green fluorescent protein; TE, trophectoderm.
homozygous for a gene trap vector in the ROSA26 locus [Gt(ROSA)26 Sor] (20) were mated with female ICR mice to generate embryos with ubiquitous expression of β-galactosidase neomycin resistance fusion protein (Bgeo) for infection with the FG12 U6 RNAi LacZ virus. Male ICR mice hemizygous for the Z/RED transgene [Tg(ACTB-Bgeo, DsRed.MST)1Nagy] (21) were mated with female ICR mice to generate embryos for infection with the Cre-expressing virus. Expanded blastocysts had the zona removed by acid Tyrode’s (Sigma, St. Louis, MO, USA) treatment (19) prior to viral infection. All embryo culture was done in microdrops of KSOM media with amino-acids (KSOM-AA) overlaid with embryo-tested light mineral oil (both from Specialty Media; Millipore).
Infection of Embryos A detailed protocol of viral infection methods is provided in the supplementary materials available online at www.BioTechniques.com. Virus preparations were diluted with CO 2 equilibrated KSOM-AA (19) to the desired titer. One microliter of diluted virus preparation was added to 9-μL droplets under oil containing up to 40 expanded blastocysts. The embryos were cultured with virus for the indicated times, then washed by serially transferring using a sequencing pipet (Drummond Scientific, Broomall, PA, USA) into droplets of at least 7 volumes clean media [0.5–1 mL M2 or Dulbecco’s modified Eagle’s medium (DMEM) plus HEPES]. Embryos were trans-
Figure 1. Trophoblast-specific enhanced green fluorescent protein (EGFP) expression after infection of wild-type blastocysts with a lentivirus containing sequences coding for an EGFP transgene (FUGW UbiC EGFP). E3.5 wild-type (ICR) blastocyst infected for 6 h with FUGW UbiC eGFP, GFP (green) expression is restricted in the trophectoderm and is not in the inner cell mass (ICM). (A–D) GFP views (green) superimposed on bright-field images of whole embryos (A, B, and D) and of placental slice, 2- to 3-mm-thick and perpendicular to the flat (fetal) side of the placenta (C). Blastocysts treated as described in panel A were transferred and dissected at E5.5 (B) and E15.5 (D). (B) E5.5 conceptus showed continued expression of EGFP in trophoblast cell derivatives: ectoplacental cone (Epc), extraembryonic ectoderm (Exe), but not in the embryo (Emb) or the visveral endoderm (arrowheads). (C) E15.5 mouse placenta, GFP expression confined within trophoblast cell lineages including giant cells (GC), spongiotrophoblast (Sp), and labyrinth (La). The weaker EGFP expression in labyrinth is due to contribution of GFP negative fetal cells. Allantoic vasculature (Al) is GFP negative, as it is entirely of fetal origin. (D) E15.5 embryo belonging to placenta shown in panel C and is GFP negative, indicating no viral infection of embryonic lineage. 318 ı BioTechniques ı www.biotechniques.com
Vol. 42 ı No. 3 ı 2007
Short Technical Reports ferred into day 2.5 pseudopregnant females as published (19) and dissected at the indicated time points.
Non-infected
Figure 2. Trophoblast-specific green fluorescent protein (GFP) expression and gene knockdown after infection of blastocysts with a lentivirus containing sequences coding for both an GFP transgene and a short hairpin RNA (shRNA) against the β-galactosidase (lacZ) gene (FG12U6RNAiLacZ). Panels A, D, I, and L, are bright-field views of non-infected whole conceptuses stained for β-galactosidase expression at the stages indicated. Panels B, E, J, and M are bright-field views of infected whole conceptuses stained for β-galactosidase expression at the stages indicated. Panel O is a bright-field view of an E18.5 non-infected placental slice, 2- to 3-mm-thick and perpendicular to the flat (fetal) side of the placenta, stained for β-galactosidase expression. Panel P is a bright-field view of an E18.5 infected placental slice, 2- to 3-mm-thick and perpendicular to the flat (fetal) side of the placenta, stained for β-galactosidase expression. Panels C, F, K, N, and Q are GFP views superimposed on their corresponding bright-field views (B, E, J, M, and P, respectively). Trophoblast-specific GFP expression and LacZ knockdown is observed from the earliest stage analyzed (A–C) and is very prominent in blastocysts cultured in vitro until 48 h after their initial encounter with lentivirus-containing medium (I–K). In contrast, their non-infected counterparts show strong LacZ expression (A, D, and I) and no GFP signal (data not shown). This trophoblast-specific GFP expression and LacZ knockdown is maintained upon in vivo development of infected blastocysts to stage E5.5 (M and N) and even until one day before birth, at the E18.5 stage (P and Q). This was not observed in the fetuses of these E18.5 placentae (ubiquitously expressing LacZ with no enhanced GFP, or EGFP, signal, data not shown). Scale bars A–K, 20 μm; L–N, 50 μm; and O–Q, 1 mm.
Infected
12 h
Visualization of Gene Expression Blastocysts to E5.5 stage conceptuses were imaged with a Leica 10× PH1 objective (NPlan, 0.25) on a Leica Inverted DMIRBE (all from Leica Microsystems GmbH, Wetzlar, Germany) using appropriate filters for GFP or DsRed. Fluorescent and bright field or Nomarski images were acquired using an Orca C4742–95 Digital Camera (Hamamatsu, Bridgewater, NJ, USA) as monochrome images and imported into Openlab software (Improvision, Coventry, UK). Some images were obtained directly as color images using an AxioCam MRC 5 and acquired with AxioVision 4.4 software (both from Carl Zeiss, Jena, Germany). E6.5 conceptuses were imaged with 1.0× objective lens with variable magnification steps on a Leica Dissecting MZ16FA microscope (Leica Microsystems GmbH). Both fluorescence and bright field images were acquired with a Retiga 1300C camera (Q-Imaging, Burnaby, BC, Canada) and imported into Openlab software. Some blastocysts were imaged by confocal microscopy. Blastocysts were mounted onto glass slides in phosphate-buffered saline (PBS) and Molecular Probes spacer (Invitrogen,
24 h
48 h
E5.5
E18.5 lacZ
lacZ
lacZ-GFP
Carlsbad, CA, USA), then imaged with a Zeiss 40×/1.2 W Koor objective (C-Apochromat®) on LSM 510 Meta, acquired with LSM 510 software (all
Table 2. Implantation, Survival, and Infection Rates After 6- or 18-h Exposure Times to FUGW Embryos (No.) Infection Time (h)
GFP Expression
Viral Titer
Transferred
Implant Sites
Normal
Delayed
Resorbed
Total Scoreda
Strong
Medium
Weak
Negative
6
0
24
22
15
0
7
15
0
0
0
15
6
9.6 × 105
49
31
28
0
1
24
0
3
1
20
6
4.8 ×
106
41
35
31
1
0
30
7
4
8
11
6
2.4 × 107
59
44
37
5
2
37
24
5
8
0
18
0
28
28
18
0
10
10
0
0
0
10
18
9.6 × 105
40
29
14
4
11
14
3
2
1
8
18
4.8 × 106
40
30
14
5
11
14
6
2
0
6
18
2.4 ×
40
19
12
5
2
12
9
3
0
0
107
GFP, green fluorescent protein. aSome embryos lost during processing.
320 ı BioTechniques ı www.biotechniques.com
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Short Technical Reports Table 3. Implantation, Survival, Infection Rates, and Knockdown Specificity of FG12 LacZ RNAi Exposure Embryos (No.) Embryonic Stage (days)
Viral Titer
Transferred
Implant Sites
Normal
Resorbed
GFP Troph Specific
LacZ Troph Negative
5.5
0
37
34
28
6
0
0
12.5
0
10
2
1
1
0
0
18.5
0
10
4
4
0
0
0
57
33
33
7
0
0
5.5
1×
107
104
22
17
5
17
17
12.5
1 × 107
24
3
2
1
2
2
18.5
1×
24
14
13
1
13
13
152
39
32
7
32
32
Total
107
Total
RNAi, RNA interference; GFP, green fluorescent protein.
A
A��
A�
*
*
* 20 �m
B
n=4
D
B��
D��
Epc Exe
Epc Exe Emb
30 �m n = 2
C
GC
C�� Epc Exe
50 �m n = 11
E
RESULTS AND DISCUSSION
F Epc
Epc
Exe Exe
Emb 30 �m n = 9
50 �m
50 �m
Figure 3. Trophectoderm-specific Cre-mediated transgene excision after infection of Z/RED embryos with EF.CRE-CMV.GFP lentivirus. (A) Confocal images of E3.5 Z/RED blastocysts infected 18 h with EF.CRE-CMV.GFP lentivirus and subsequently cultured for 48 h. Only trophectodermal cells were infected with lentivirus as indicated by enhanced green fluorescent protein (EGFP) expression (green, A′). Successful Cre-mediated excision of loxP flanking lacZ sequence in trophectodermal cells of Z/RED embryos is indicated by the presence of red fluorescence (A″). (B–D) E3.5 Z/RED blastocysts were transferred and dissected at E5.5 (B and C) and E6.5 (D). Cre-mediated specific excision of trophoblast cell derivatives indicated by DsRed expression is apparent in B″–D″. Bright-field (B, C, and D) and DsRed (B″, C″, and D″), respectively. Ectoplacental cone (Epc), extraembryonic ectoderm (Exe), and primary giant cell (GC) were all DsRed positive (B″–D″). (E and F) Sections of 5-bromo-4-chloro3-indolyl-β-d-galactopyranoside (X-gal) stained non-infected E6.5 conceptus control (E) and conceptus infected with the highest titer of EF.CRE-CMV.GFP lentivirus [2.4 × 107 infectious units (ifu)/mL] (F) showing loss of LacZ stain in the Exe and Epc (E and F, arrowheads). Emb, embryo proper (epiblastderived tissues). 322 ı BioTechniques ı www.biotechniques.com
from Carl Zeiss). The pinhole was set to 1–1.2 airy unit, and a series of optical sections was taken for all samples. LacZ staining of the Z/RED and ROSA26 embryos was as previously described (22). Some lacZ-stained embryos were paraffin-embedded and sectioned according to standard histological protocols. Sections were imaged with a Zeiss 10× PH1 objective (PlanNeofluar 0.30) on an Axioskop (all from Carl Zeiss), using an AxioCam HRc, and acquired with AxioVision 4.4 software. All images were then compiled into figures using Adobe® Photoshop® and Illustrator® CS2.
Using the FUGW vector, in which GFP is driven by the constitutive ubiquitin C promoter (11), we infected fully expanded E3.5 mouse blastocysts with and without the zona in a suspension of viral particles in embryo culture medium for 4–18 h. Embryos were then washed through multiple changes of medium to remove excess viral particles and cultured further. GFP expression was first observed 4 h after infection in zona-free embryos only, and by 24 h from the start of infection, all zona-free embryos showed GFP expression (Table 1). Removal of the zona is critical to viral infection. Expression was clearly restricted to the outer TE cells, and not the ICM, and was widespread if not ubiquitous in the TE layer (Figure 1A). Different sets of Vol. 42 ı No. 3 ı 2007
Short Technical Reports blastocysts exposed to FUGW virus for 6 h were transferred directly to pseudopregnant recipients and dissected out at different stages of development. At all developmental stages examined, all conceptuses recovered showed GFP expression restricted to the trophoblast derivatives; no expression was ever seen in the derivatives of the ICM of the blastocyst (Figure 1, B and D). In most cases, expression was seen throughout the trophoblast lineage, even in placentae dissected close to term (Figure 1C). Thus, lentivirusdriven transgenes can be expressed without apparent silencing in the TE and its trophoblast derivatives and can act as lineage tracers for TE derivatives. We then tested postimplantation survival and expression after infection for different times and at different viral titers. The results (Table 2) showed that more embryos and more trophoblast cells per embryo were infected when either the viral titer was increased or the incubation period was increased from 6 h to overnight, without any cross-infection of the ICM. There was no clear evidence for any toxicity effect related to higher viral titers. There was some increased embryo delay and loss in the overnight-infected embryos compared with those incubated for 6 h and transferred the same day. However, this same difference was also seen in control embryos cultured overnight before transfer. A shorter infection time with high viral titer may increase postimplantation survival and alleviate embryo delay, but a longer infection period can result in more uniform trophoblast expression. Viral titer and incubation period can be varied at will to achieve appropriate levels of
blast lineages (Figure 2, N and Q) and a major reduction in lacZ expression in the same lineages (Figure 2, L and M) when compared with controls (Figure 2, O and P). This demonstrates proofof-principle that lentiviral infection can be used for rapid in vivo RNAi-based functional screens for trophoblast gene pathways, complementing recent demonstrations of the use of RNAi screens in ES cells (24) and ES cellderived embryos (25) to explore gene function in the embryonic lineages. True loss of function analysis in the trophoblast lineage requires trophoblastspecific gene knockout, and so we also generated a lentivirus vector expressing the Cre recombinase. We utilized a third viral vector with a dual promoter (16), such that Cre was expressed from the elongation factor1α (EF) promoter and GFP was expressed from the cytomegalovirus (CMV) promoter (EF.Cre-CMV. GFP). This generates two transcripts, one from the EF promoter with Cre in frame followed by a stop codon and GFP out of frame, while the second is from the CMV promoter with only GFP in frame. Both transcripts share the same poly(A) signal. The CMV.GFP is flanked by loxP sites so that upon genomic integration and expression of Cre, the GFP signal will be lost. Any GFP signal remaining is indicative of poor excision, while loss of GFP can be measured in a time course to monitor the rate of excision. Future versions of this vector will have the loxP sites removed, so that GFP will be a marker of viral infection at all stages of development. Heterozygous males from the Cre reporter line, Z/RED or Tg(ACTB-Bgeo, DsRed.MST)1Nagy (21), were crossed with wild-type females, and blastocysts
transgene expression in the trophoblast lineage. Lentivirus-mediated transgene expression can thus be used for gain of function and rescue of function experiments in the trophoblast lineage at all stages. We were interested in also developing methodology to produce loss of gene function in the trophoblast. We and others have shown that pol IIIdriven short hairpin RNAs (shRNA) can be introduced into the embryonic lineages via ES cells and can effectively down-regulate gene expression in vivo (23). The effectiveness of pol III-driven transgenes in suppressing gene expression in the trophoblast has not been assessed. The FG12 vector carrying the GFP transgene along with a pol III-driven shRNA directed against the Escherichia coli β-galactosidase (lacZ) gene (14) was used to infect blastocysts homozygous for the ubiquitously expressing ROSA26 gene trap insertion (20). Blastocysts were infected for 6 h at the same viral titer used for the most effective FUGW infections, cultured in vitro, observed for GFP expression, and then fixed and stained for lacZ expression. By 12 h, when GFP was clearly seen in the TE cells, the expression of lacZ was already lower in the TE (Figure 2B) than in uninfected controls (Figure 2A). Given the known perdurance of lacZ protein, this suggests a very rapid effect of the lentiviral RNAi. By 48 h, lacZ expression was apparently absent in the TE of the infected blastocysts (Figure 2, E and J). Some blastocysts were transferred to recipients and allowed to develop to different stages (Table 3). All dissected conceptuses (n = 22, E5.5; n = 13, E18.5) showed strong GFP expression throughout the tropho-
Table 4. Implantation, Survival, Infection Rates, and Cre-Excision Specificity After EF.Cre.CMV.GFP Viral Vector Exposure Embryos (No.)
Z/RED Transgene Positive Resorbed Embryosa
Transferred
Implants
Normal
Delayed
105
63
52
30
2
20
19
4.8 × 106
66
45
37
0
8
17
2.4 ×
72
30
23
0
7
11
Viral Titer 9.6 ×
107
RFP Positive
LacZ Positive LacZ ExE Positive
Medium
Weak
LacZ ExE Negative
0
1
2
0
19
0
11
3
5
12
6
5
0
11
0
Strong
RFP, red fluorescent protein. aSome embryos lost during processing.
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www.biotechniques.com ı BioTechniques ı 323
Short Technical Reports were infected with the Cre lentivirus as before. In this reporter line, expression of Cre inactivates lacZ expression, via excision of the loxP-flanked sequences, and allows expression of a DsRed transgene (21). After 24 h in culture, some TE cells began to express DsRed, indicative of Cre excision of the reporter transgene, and, by 48 h, colocalized expression of DsRed and reduced GFP indicated effective excision in many TE cells (Figure 3, A′ and A″). After transfer to the uterus and dissection at 5.5 days (Figure 3, B, B″, C, and C″) and 6.5 days (Figure 3, D and D″) of development, DsRed expression was observed in transgenic conceptuses in trophoblast derivatives only (Table 4). At the highest titer of virus (2.4 × 107 U), 11/11 E6.5 conceptuses expressed DsRed trophoblast specifically and showed complete loss of β-galactosidase expression in this lineage (Figure 3, E and F). At lower titers, some persistent lacZ expression was seen in the trophoblast, demonstrating mosaic excision (data not shown). GFP expression from this particular vector was no longer observed at all after implantation due to the excision of the CMV.GFP cassette, which is flanked by loxP sites. Given that GFP expression was observed in blastocysts, this suggests that excision is complete in all cells that were infected with the virus, and any mosaicism observed in lacZ expression is a result of mosaic infection. Embryos infected at high titers showed widespread DsRed expression in the trophoblast alone (Figure 3D″), demonstrating that this can be an effective method for rapid generation of trophoblast-specific gene knockouts. Since excision begins at the blastocyst, this approach provides the best method to date to assess gene function in trophoblast versus embryonic lineages from their separation at the blastocyst stage. Use of an inducible Cre, such as the tamoxiphen-inducible CreERT, in the vectors would allow conditional knockout at different times in development to further explore trophoblast gene function. By simple control of the time of lentivirus infection during preimplantation development, we have developed a suite of tools for both gain and loss of function analysis of gene function 324 ı BioTechniques ı www.biotechniques.com
in the trophoblast lineage, a key component of embryonic patterning and placental development. The remarkable efficiency of infection and expression of lentivirus-mediated transgenes makes this approach a feasible means of rapid in vivo screening for putative trophoblast gene regulators and separation of gene functions between embryo and extraembryonic lineages. Depending on viral titer and length of infection period, expression can vary from apparently uniform expression in all cells to clear mosaic expression. Generating a set of conceptuses with different levels of expression of the genetic mediator of choice can be a useful way of generating a phenotypic series and examining the behavior of genetically modified trophoblast cells in competition with wild-type cells. Since the vectors all contain reporter transgenes, it is easy to monitor mosaicism and correlate gene modification with cellular phenotype. Pseudotyped lentiviruses can infect across species barriers, so this method could also be used to compare gene function in the placenta in different mammalian species. ACKNOWLEDGMENTS
We thank Drs. Carlos Lois and XiaoFeng Qin for the FUGW and FG15 vectors, respectively, and Eric Sat for viral preparations. This work was supported by funds from the Canadian Institutes of Health Research (CIHR). B.C. is supported by a CIHR Doctoral Fellowship, and J.R. is supported by a CIHR Distinguished Scientist Award. K.C. holds a C.J. Martin Fellowship. COMPETING INTERESTS STATEMENT
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Received 17 October 2006; accepted 10 November 2006. Address correspondence to Pantelis Georgiades, Developmental Genetics and Embryology Research Unit, Department of Biological Sciences, University of Cyprus, New University Campus, 75 Kallipoleos Avenue, P.O. Box 20537, 1678 Nicosia, Cyprus. e-mail:
[email protected]; or Janet Rossant, Program in Developmental and Stem Cell Biology, Research Institute, Hospital for Sick Children, Toronto, ON, Canada. e-mail:
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