Inducible and selective transgene expression in murine vascular endothelium

Peter I. Teng1,2, Maria R. Dichiara1, László G. Kömüves1, Keith Abe1, Thomas Quertermous2 and James N. Topper1,2

1 Millennium Pharmaceuticals, South San Francisco 94080
2 Division of Cardiovascular Medicine, Stanford University Hospital and Clinics, Stanford, California 94305


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
We have developed a system utilizing the murine Tie2 promoter/enhancer coupled with the "tetracycline-on" regulatory elements to create a model that allows regulated and selective expression of a ß-galactosidase (ßGal) reporter transgene in the adult murine vascular endothelium. Two independent lines of viable and fertile mice were characterized, and they exhibit minimal ßGal expression under basal conditions. In response to exogenous doxycycline (Dox), selective expression of ßGal was demonstrated in the vascular endothelium of all tissues examined. En face analyses of the aorta and its principle branches indicate that the vast majority of lumenal endothelial cells express the transgene. Inducible ßGal expression also extends to the endocardium and the microvasculature of all organs. There is no evidence of specific transgene expression in nonendothelial cell types. Induction of the ßGal was effectively achieved after 3 days of oral Dox treatment and persisted for over 3 mo with continuous administration. This model can now be widely applied to study the role of specific genes in the phenotype of adult murine vasculature.

transgenic mice; Tie2; tetracycline regulation system; gene expression; vascular biology


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
TRANSGENIC TECHNOLOGY has afforded us the ability to overexpress specific target genes in mice. These approaches have been significantly enhanced by the use of tissue- or cell-type-selective promoters which allows one to target a specific tissue or cell type of interest. In the cardiovascular system, such systems have been widely used to selectively manipulate the expression of genes in both the heart and vasculature (3, 4, 9, 13, 28). However, because of the central roles the heart and vasculature play during normal development, these studies are often accompanied by developmental defects that limit their application and relevance to adult disease state. This is particularly true for the vascular endothelium, as evidenced by the numerous transgenic mice involving endothelial-expressed genes that have been reported to harbor severe developmental abnormalities (1, 4, 9, 18). For this reason, several systems that employ transcriptional switches to allow temporally regulated control of expression have been developed. One such system known as the tetracycline-inducible system was originally described and developed by Bujard and colleagues (14, 16, 17, 20), Schultze et al. (37), and Shockett et al. (38, 39). In this system, a modified form of the tetracycline-responsive transactivator protein derived from bacteria is overexpressed via a tissue- or cell-type-selective promoter. This transactivator protein then modulates transcription of a coexpressed transgene harboring a promoter that is responsive to this transactivator protein in the presence (or absence) of exogenous tetracycline. Thus the tissue- or cell-type-selective promoter provides regional selectivity, and the addition (or removal) of exogenous tetracycline provides temporal control. This methodology has been adapted in forms known as the "tetracycline-on" (Tet-on) and "tetracycline-off" (Tet-off) regulatory systems. In the former, the transactivator protein has been engineered to activate transcription in the presence of the tetracycline [or its derivative, e.g., doxycycline (Dox)], whereas in the latter, the transactivator represses transcription in the presence of the drug ligand. In both cases, action of the transactivator is highly selective to promoters containing only tetracycline response elements. Both strategies have been adapted by numerous investigators in both cell-based experiments and in vivo animal models (5, 26, 30, 41, 43).

A number of promoters have been reported to be capable of directing endothelial-selective expression in vivo. These include promoters from the tek/Tie2, CD31, ICAM-2, von Willebrand factor (vWF), FLT-1, pre-proendothelin (P-PET), and E-selectin genes (2, 6, 19, 28, 31, 36, 42). Among these, the available literature suggests that the Tie2 promoter is among the most effective in its ability to selectively direct expression of exogenous genes to the majority of endothelial cells in vivo (10, 11, 35). The tek/Tie2 gene encodes the receptor for angiopoietin and is a member of the receptor tyrosine kinase family. Its expression can be detected in early embryonic endothelial cells and persists throughout adult vascular development (8, 11, 33, 34, 35). To develop a system that would allow investigators to selectively manipulate the expression of genes in vascular endothelium in vivo, we have coupled the Tet-on system to the Tie2 promoter and its enhancer intron segment. This system is designed to allow regulated, selective overexpression of virtually any transgene in murine vascular endothelium in vivo.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Construction of Tie2-rTA and TRE-ßGal transgenes.
The tek/Tie2 promoter and its enhancer intron was provided by Dr. Bahaa Fadel (11). The 2.1-kb tek/Tie2 promoter fragment spans 1.8 kb of the Tie2 upstream regulatory sequence and 318 bp of 5'-untranslated region in the first exon (Fig. 1A). It was excised from its original pGL2 basic vector using HindIII on both ends and subcloned into the pKS Bluescript vector to create pKS Tie2. The enhancer element used consisted of the 1.7-kb XhoI/KpnI fragment from the first intron originally sequenced and identified by Schlaeger et al. (35) (this appears to be necessary to enhance Tie2 promoter activity in adult murine endothelium). This intron fragment was subcloned into a separate pKS Bluescript vector to create pKS intron, then subsequently subcloned into pKS Tie2 to create pKS Tie2/intron. The pTet-On vector (K1621-A; Clontech, Palo Alto, CA), was digested with EcoRI/PvuII to obtain the 1.6-kb rTA gene together with the SV40 polyadenylation sequence and subcloned into psp72. This combination sequence was then excised from psp72 with XhoI/ClaI to provide matching digestion sites and subcloned into the pKS Tie2/intron to achieve the final pKS Tie2-rTA vector. The nuclear-localizing ß-galactosidase (nl-ßGal), along with its own poly-A signal (3.5 kb), was excised from pnLacF vector (provided by Dr. David Milstone) with XbaI/HindIII and subcloned into pTRE (K1620-1; Clontech), between nucleotide sequences 477 and 941, to create the pTRE-ßGal vector.



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Fig. 1. Genetic characterization of the Tie2-rTA/TRE-ßGal transgenic mouse. A: schematic representation of the Tie2-rTA and TRE-ßGal constructs used to generate transgenic mice. The Tie2-rTA construct consists of the Tie2 promoter (2.1-kb HindIII/HindIII segment spanning nucleotides -1800 to +318, with respect to the start of transcription) and the 1.7-kb enhancer segment (XhoI/KpnI) derived from the first 10-kb intron, together with the gene coding for the Tet-on transactivator (rTA) (1.6-kb EcoRI/PvuII segment spanning nucleotides 767 to 2400 from the pTet-On vector; Clontech). The TRE-ßGal construct contains the ß-galactosidase (ßGal) sequence with a nuclear-localization ("nl") motif (3.5 kb) with its own polyadenylation signal inserted (between XbaI/HindIII) downstream to the tetracycline (Tet)-responsive element (TRE), which consists of a minimal promoter and seven operator sequences (tetO) from the pTRE vector (Clontech). B: genotype confirmation of F1 generation Tie2-rTA/TRE-ßGal transgenic lines by Southern blotting. Less than 10 copies of the transgenes were incorporated into the mouse genome (left vs. right). Line 1425 shows a lower gene copy than line 1420 (left). C: frequency of transgenic offspring obtained over 4–6 generations of backcrosses. The ratio represents the number of transgenic offspring to their litter. The near 50% transgenic transmission rate in both the 1425 and 1420 lines supports germ line transmission without embryonic lethality from either Tie2-rTA or TRE-ßGal constructs.

 
Generation of transgenic mice.
All procedures and protocols were approved by the Institutional Animal Care Research Advisory Committee at both Stanford University Medical Center (Stanford, CA) and COR Therapeutics (now part of Millennium Pharmaceuticals, South San Francisco, CA). The 5.4-kb Tie2-rTA transgene was excised with NotI/Asp718, and the 3.9-kb TRE-ßGal transgene was excised with XhoI/HindIII, from their respective vector backbones (Fig. 1A). These transgenes were gel-purified and extracted with GeneClean kit (Promega, Madison, WI) and comicroinjected into fertilized C57BL/6 x CBA oocytes. The genotype of offsprings was identified by both Southern blotting and PCR of tail DNA extracted using DNeasy Tissue Kit (Qiagen, Valencia, CA). PCR genotyping was used for subsequent generations. The following primers were used for amplification of the Tie2-rTA sequence: forward, 5'-GCT CGC ATG GTC CAC TCG-3', and reverse, 5'-GCA AAA GTG AGT AG GTG CC-3', which resulted in a product size of 260 bp. The TRE-ßGal primers were as follows: forward, 5'-GGC GTG TAC GGT GGG AGG-3', and reverse, 5'-CGG GAT CCC CCA TGC TCC CC-3', with product size 270 bp. The PCR reaction included 1x PCR buffer (Perkin-Elmer), 200 µM of each nucleotide, 0.2 µM of each primer pair, 2 mM MgCl2, and 0.6 U of HotStarTaq DNA Polymerase (Qiagen) per reaction, activated for 10 min at 95°C, followed by 32 cycles of 95°C for 1 min, 62°C for 1 min, and 72°C for 1.5 min, with a final extension step at 72°C for 10 min. For Southern blotting, a 1,025-bp fragment of the rTA from the Tet-on vector, digested with EcoRI/BamHI, was utilized. The pKS Tie2-rTA vector was used as a positive control for estimation of transgene copy number.

Transgene induction protocol and tissue harvest.
Four routes of Dox administration were compared with no Dox (i.e., saline controls) for the induction of the reporter transgene. Four sets of transgenic littermates in groups of three were treated separately with oral Dox-containing chow (200 mg/kg; Bio-Serv, Frenchtown, NJ), Dox-containing water (2 mg/ml; Sigma Chemicals, with 5% sucrose added) ad libitum, Dox-containing water given through oral gavage (6 mg/ml, 0.5 ml/gavage, twice daily), or Dox-containing saline given through intraperitoneal injection (12 mg/ml, 0.25 ml/injectate, twice daily). These different routes provided a range of ~1–6 mg of Dox per day. For most experiments, oral Dox was administered in the drinking water or as Dox-containing chow. For analysis of transgene induction during development, Dox was administered to transgenic mothers on day 1 postcoitus (determined by vaginal plug examination) and maintained for the duration of the gestation period before embryo harvest on day 9.5 or 17, respectively. During harvest, the mother was perfused as described below, and the uterus was dissected out to remove each individual embryo within the yolk sac.

For tissue analysis, mice were anesthetized with a ketamine/xylazine mixture (intraperitoneally) prior to tissue and vessel harvest. Each animal was pressure-perfused initially with 5 ml cold saline (at constant rate of 5 ml/min), followed by 10 ml of cold fixative solution (0.2% glutaraldehyde/PBS), delivered through the left ventricle with an exit cut in the right atrium. Organs of interest (e.g., aorta, heart, lung, kidney, liver, brain, spleen) were subsequently removed and placed in the same fixative solution. Bone marrow smears were prepared on charged glass slides from femur. Marrow was extruded and air dried.

Histochemistry: X-Gal staining.
Organs and vessels harvested from the mice were fixed for an additional 60–90 min in 0.2% glutaraldehyde/PBS on ice and then rinsed with three washes of a solution of PBS containing 0.01% sodium deoxycholate, 2 mM MgCl2, and 0.02% Nonidet P-40 (NP-40) for 15 min each, followed by overnight staining (12–16 h at 32–37°C) with X-Gal solution [0.01% sodium deoxycholate, 2 mM MgCl2, 0.02% NP-40, 20 mM Tris-Cl, pH 7.3, 1 mg/ml 5-bromo-4-chloro-3-indolyl-ß,D-galactopyranoside (X-Gal), 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6]. Bone marrow smears were fixed on slides with 0.2% glutaraldehyde/PBS for 5 min, followed by brief rinse and overnight staining with X-Gal solution.

Day 9.5 embryos were stained as whole mounts, whereas day 17 embryos were bisected longitudinally before fixation, then stained in the same manner as above. After staining, tissues were fixed further in 4% paraformaldehyde/PBS and kept at 4°C before embedding in paraffin for sectioning. The en face analysis of the aorta was performed by clarifying the isolated, X-Gal-stained aorta via serial dehydration in ethanol solutions (70%, 90%, 99%), followed by brief immersion in methyl salicylate to attain clarity, then cutting and mounting the tissue on glass slides with coverslips for microscopy.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Generation of Tie2-rTA/TRE-ßGal mice.
Two independent Tie2-rTA/TRE-ßGal founder lines were identified (designated 1420 and 1425, respectively) by PCR genotyping of 37 offspring derived from coinjected zygotes. These two lines were also confirmed by Southern blotting, and the copy number of the transgene was estimated to be less than 10 in both lines (Fig. 1B). Both of these lines demonstrated reproducible transmission of the transgene to subsequent generations in a Mendelian fashion. A transmission rate of ~50% was consistently observed over six generations (Fig. 1C). Greater than 95% of the transgenic mice across all generations were confirmed to harbor both transgenes (i.e., Tie2-rTA and TRE-ßGal), indicating the close proximity of the two inserted transgenes within the mouse genome. The approximate 3–5% loss of the TRE-ßGal transgene may have been due to random deletion or recombination. Both lines of mice demonstrated endothelial-selective, inducible nl-ßGal expression. All of the expression data shown here are derived from the 1425 line, and the data from the 1420 line are qualitatively similar.

Performance of the Tie2 promoter/enhancer-based Tet-on system.
Near-uniform expression of the nl-ßGal reporter gene was achieved in the endothelial nuclei of the aorta and all its major and minor branches in response to treatment of the mice with Dox. Figure 2, A and B, represents an example of robust induction of the reporter gene in the aorta when the mouse was treated with Dox. In the absence of exogenous Dox, there was a variable amount of spotty expression of the ßGal transgene. This expression was largely confined to the smaller branches of the aorta and appeared to be due to a basal level of leakiness in the system, since this expression was also confined to endothelial nuclei. Figure 2C shows X-Gal staining restricted to endothelial nuclei on cross section of the aorta, indicative of nuclear-targeted transgene expression that is highly specific to endothelial cells and not observed in other cell types in the vasculature. Similar levels of induction were obtained in whole organ staining. Surface vessels of the brain and heart expressed the ßGal transgene uniformly in their endothelial nuclei, as did the small vasculature within the lung parenchyma (Fig. 2, GI). This pattern of staining was not seen in comparable organs from transgenic sibling animals not treated with Dox (Fig. 2, DF).



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Fig. 2. Doxycycline (Dox) treatment selectively induces the expression of the reporter transgene protein in various organs. Animals were treated ad libitum with Dox-containing drinking water (2 mg/ml) for 7 days (B and C) and for 3 mo (GI). Tissue samples were obtained from both Dox-treated (B and C, and GI) and untreated control (A, and DF) transgenic littermates, followed by X-Gal staining for ßGal activity. Induction of the transgene was seen in the thoracic aorta (A vs. B) as well as in the vasculature at the base of the brain (D vs. G); macroscopic view of the lung hilar surface (E vs. H); and macroscopic view of the heart at the level of the great vessels (F vs. I). Cross section of the X-Gal-stained thoracic aorta shows the endothelial selectivity and nuclear localization of the reporter transgene (C). Low basal level of X-Gal staining can be seen in endothelial cells of small aortic branches (A).

 
Expression of the ßGal transgene was seen after 3 days of Dox administration and persisted for at least 3 mo with continuous treatment. We observed no significant differences in the extent or time course of induction of the endothelial expressed nl-ßGal in response to distinct routes of Dox administration. The use of Dox-containing chow, and ad libitum or oral gavage of Dox-containing drinking water, yielded similar results, as did intraperitoneal injection of Dox-containing saline (data not shown). Thus it appears that the extent of reporter gene expression in this system is independent of route of administration or dose (within the range examined here) of Dox.

To further assess the extent of endothelial expression of the reporter gene after Dox induction, a comprehensive morphological and histological analysis of these mice was performed. En face analysis of the excised aorta revealed a near-uniform pattern of nl-ßGal expression in the endothelial monolayer. In fact, these cells course along tissue grooves and are visualized to aggregate distal to aortic branching points (Fig. 3, A and B). Near-uniform endothelial expression was demonstrated in all vessels examined. These include the intercostal arteries, the carotid, iliac arteries (Fig. 3, CE), and vessels of the brain and of the heart (Fig. 3, F and G). Inducible expression of nl-ßGal transgene also extended to the endocardium and the endothelial layer of cardiac valves (Figs. 4A and 3H), as well as to the microvasculature of all organs examined. These included small vessels within the parenchyma of the brain and lung (Fig. 4, B and C) and of the myocardium and kidney, including the arterioles supplying the glomerulus (Fig. 4, D and E). These results were also confirmed by CD31 staining of the endothelial cells. Marrow smears demonstrated clear endothelial nl-ßGal expression in Dox-treated mice, with no evidence of specific expression in the leukocytes. Control mice of both non-Dox-treated mice and wild type demonstrated only nonspecific X-Gal staining in the marrow (data not shown). Other organs examined, including the liver, spleen, uterus, prostate, small and large intestine, stomach, and skeletal muscles, were also all noted to express endothelial-specific nl-ßGal in response to Dox (data not shown).



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Fig. 3. Endothelial expression of the reporter transgene is seen extensively in the cardiovascular system following Dox treatment. A and B: en face analysis of aorta reveals near-uniform expression of the transgene in the lumenal endothelial monolayer. CF: Endothelial expression of nuclear-localized ßGal is observed in all blood vessels examined, including the intercostal (C), carotid (D), and iliac (E) arteries, as well as cerebral arteries, seen from the surface of the brain (F). G and H: transgene expression also extends to the intramyocardial vessels, seen on coronal section of the heart (G), and endothelial lining over cardiac valves, such as the tricuspid valves (H).

 


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Fig. 4. Histological analysis of Dox-induced reporter transgene expression in the microvasculature. Tissue samples following X-Gal staining were paraffin embedded, sectioned, and counterstained with Nuclear Fast Red. A: in the heart, reporter transgene expression is uniformly seen in the endocardium in both ventricles and over the tricuspid valves. BD: small vessels and microvessels express the transgene in the cerebrum (B), lung (C), and within cardiac muscle (D). E: renal expression of the transgene in the afferent arteriole and glomerular capillaries. Note: because of the nuclear localization of the reporter ßGal, only endothelial cells whose nuclei are contained within the section are visualized.

 
To assess the performance of the system during development, Dox was administered to pregnant females beginning 1 day after mating. Embryos were harvested, and X-Gal staining was performed as whole mounts. Nuclear-localized X-Gal staining was observed in the embryonic vasculature on day 9.5 (the earliest time point examined). At this stage, nl-ßGal expression was observed in the primitive heart and branchial arch arteries (Fig. 5A), as well as in the yolk sac vasculature. At day 17, uniform expression of nl-ßGal was seen in the aorta, intercostal arteries, arch vessels (e.g., the carotid), vascular component of ribs, and vessels supplying the heart, lung, and other organs examined (Fig. 5, B and C).



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Fig. 5. Inducible expression of the reporter transgene during embryonic and fetal development. Pregnant females were treated ad libitum with Dox-containing drinking water (2 mg/ml), beginning 24 h postcoitus and throughout gestation. Whole embryos were stained with X-gal. A: in day 9.5 embryo, positive X-Gal staining is seen in the primitive heart (ht), pre-aortic branchial arteries (ba), and vascular lining of yolk sac (ys). B and C: day 17 fetus stained after sagittal bisection shows endothelial-selective nuclear localized X-Gal staining in the coronary (cv) and pulmonary vessels (pv), as well as in the aorta (ao), branching intercostals arteries (ic), and carotid artery (ca).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Standard transgenic methods have been extremely valuable for the understanding of disease pathways. For example, these efforts have identified multiple genes in the heart and vasculature that are essential in maintaining vascular homeostasis. However, for many classes of such genes, attempts to modulate their expression within the cardiovascular system (and in particular within the vasculature itself) have caused severe developmental defects up to and including embryonic lethality (7, 15). For example, the actions of growth factors such as PDGF and TGF-ß (and the components of their respective signaling pathways) have been strongly implicated in human vascular pathogenesis (22, 23, 24, 25, 27, 29). Because these same factors play such an essential role in normal embryonic development, most attempts to utilize transgenic technology to alter their expression or actions in vivo have resulted in severe developmental abnormalities (23, 40). These effects limit one’s ability to utilize transgenic approaches to understand the roles of these genes in the adult within the context of acquired vascular disease. For this reason, we sought to develop a system that would allow regulated transgene expression, specifically targeted to the vascular endothelium. The Tet-on system, coupled with the Tie2 promoter/enhancer, was adapted for this purpose. The Tet-on system was chosen because we predicted many of the transgenes that we and others would choose to overexpress, such as dominant-negative or constitutively active signaling molecules, would cause adverse effects during development. In this context, the Tet-on system allows one to select for viable and grossly normal appearing mice in the absence of Dox, a situation where only lines of mice manifesting either no expression or a minimal level of basal transgene expression would emerge. The Tet-on approach can then offer a rapid onset of transgene induction by the administration of Dox. Other investigators have demonstrated that this induction can also be reversed upon cessation of Dox (5, 41, 43). Thus this system may allow additional versatility in experimental design over alternate approaches such as the CRE/Lox system.

The Tie2 promoter and enhancer were chosen because of previous work demonstrating their efficacy in directing expression of reporter genes exclusively to the vascular endothelium (11, 35). In our hands, the use of the promoter region together with the 1.7-kb segment from the first intron was sufficient to attain robust endothelial expression in both the adult and during embryonic development. Also, we chose to coinject both the Tie2-rTA construct and the TRE-nl-ßGal reporter construct to facilitate screening of the system without the requirement for extensive intercrosses. Thus the low and variable level of basal ßGal expression ("leakiness") that we observed in these mice may, in part, be due to the proximity of the nl-ßGal reporter transgene to the Tie2 promoter cassette. In addition, as a result of this coinjection strategy, the use of these strains of mice to overexpress additional transgenes by crossing to distinct lines harboring the TRE cassette will necessarily involve nl-ßGal coexpression in the endothelium. Although we have not observed any adverse effects of prolonged ßGal expression (~3 mo), the effects of its coexpression with other transgenes is unknown at this time.

These efforts resulted in the creation of two independent lines of mice that demonstrate robust and selective inducible expression of a reporter transgene in the vascular endothelium. In the absence of exogenous Dox, X-Gal staining of tissues derived from both lines of mice did reveal a variable level of nuclear staining in some endothelial cells. This was typically seen in a minority of endothelial cells and was largely confined to a subset of smaller arterial branches off the major arteries. In contrast, in response to Dox, nl-ßGal expression was consistently observed to reach a significantly greater and more uniform level, in all vessels regardless of caliber, and in all vascular tissues examined. Detailed en face analysis of the aorta revealed that most of the endothelial cells of this major vessel were expressing the transgene, and a comprehensive tissue survey revealed that the induced transgene could be demonstrated in virtually all vascular organ beds, in both arteries and veins, and not observed in nonendothelial cell types. Thus we have successfully created a system that can direct the controlled induction of an exogenous transgene to the vascular endothelium in an adult mouse. Although another group of investigators has generated similar "binary transgenics" using the Tet-off approach, the Tie2 promoter they used lacked the intron enhancer element, and directed transgene expression only to the embryonic endothelium (32). We did not observe any significant or reproducible nl-ßGal expression in leukocytes. A recent report demonstrated the ability of Tie2-CRE transgenic mice to excise a "loxed" target gene in some leukocyte subsets (21). The discrepancy between these results and those reported here may be due to either very low or transient activity of the Tie2 promoter in some subsets of leukocytes. This pattern of promoter activity could have been sufficient to allow CRE-mediated excision of the target gene in these cells but may not be detectable as inducible, stable nl-ßGal expression in the adult marrow.

Interestingly, the original lines of mice were created in a mixed C57BL/6 x CBA genetic background and subsequently backcrossed to the C57BL/6 background. Although the performance of the system (as assessed by the degree of inducibility and robustness of expression) did not change initially, we found a trend toward diminished reporter transgene expression in the system as the number of backcrosses progressed. This trend was reversed by recrossing the mice back into the CBA strain, thus recapitulating the original mixed genetic background. The reasons for this observation are not clear. However, a similar phenomenon has been observed by other investigators, who found that both transactivator mRNA levels and reporter transgene activity decreased over time in animals backcrossed into the NMRI background (12).

The endothelium plays a critical role in the regulation and maintenance of vascular homeostasis. In the adult, alterations in endothelial phenotype in response to both humoral (e.g., hypercholesterolemia) and biomechanical (e.g., hypertension) stimuli are thought to play a major role in the pathogenesis of vascular diseases such as atherosclerosis. These phenotypic changes occur as a result of alterations in the expression of endothelial genes, many of which have now been identified. The development of a robust system to allow one to selectively manipulate the expression of these genes in the vascular endothelium in vivo should provide an important opportunity to dissect the role of individual genes and signaling pathways in endothelial cell biology.

To our knowledge, this is the first published report of the successful use of an endothelial cell-specific promoter utilizing the Tet-on system for gene targeting in the vasculature of an adult mouse. We believe these mice will provide a valuable system for the manipulation of endothelial gene expression in vivo.


    ACKNOWLEDGMENTS
 
We are indebted to Drs. Bahaa M. Fadel and Stephane C. Boutet for assistance with the Tie2 promoter construct; Dr. David Milstone for providing the pnLacF vector; Yan-ru Chen and the Stanford transgenic core for assistance with the microinjections; Francis Deguzman and Eduardo Escobar for assistance with tissue harvest and animal husbandry; and Scott Wasserman and Jim Tomlinson for editorial input.

This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Vascular Training Grant Award 5T32-HL-07708 (to P. I. Teng); and by NHLBI Award HL-62823 and a Howard Hughes Junior Faculty award (to J. N. Topper).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: J. N. Topper, Millennium Pharmaceuticals, Inc., 256 E. Grand Ave., South San Francisco, CA 94080 (E-mail: jamie.topper{at}mpi.com).

10.1152/physiolgenomics.00059.2002.


    References
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 

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