gfap transgenic mice

METHODS: A Companion to Methods in Enzymology 10, 351–364 (1996)

Article No. 0113

GFAP Transgenic MiceMichael Brenner* and Albee Messing†*Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health,Bethesda, Maryland 20892-4128; and †Department of Pathobiological Sciences, School of VeterinaryMedicine, University of Wisconsin-Madison, Madison, Wisconsin 53706

tein levels are controlled primarily through transcrip-The ability to direct expression of genes to astrocytes in mice tional regulation of its encoding gene (4, 5, reviewed

has been one of the major motivators of transcriptional analyses in 6). Consequently, understanding the mechanism ofof the glial fibrillary acidic protein (GFAP) gene. Another has GFAP gene regulation is of interest for insights intobeen the possibility of discovering signaling pathways that oper- astrocyte development and function and for under-ate during development, disease, and injury—all states that standing the brain’s response to injury. In addition, ifincrease GFAP gene activity—by identifying and working back the regions of the GFAP gene responsible for its cellfrom the responsible DNA elements. Here we review studies in specificity can be isolated, it becomes possible to directboth these areas and provide practical guidelines for the con- the expression of other genes in astrocytes to teststruction and analysis of GFAP transgenes. Analyses of the GFAP hypotheses of astrocyte function, manipulate the CNSpromoter from cell transfection experiments are summarized to

environment, and produce models of disease.provide background information for the studies in transgenics.This review describes studies carried out in trans-Another section provides practical information on the construc-

genic mice to identify GFAP regulatory regions andtion and analysis of transgenic mice, with particular reference toalso summarizes results obtained by using the pro-GFAP transgenes. The survey of analyses of GFAP transcriptionmoter to investigate the biological effects of variouselements in transgenic mice reveals that a segment of about 2transgenes. Studies carried out in vitro or in culturedkb of the 5*-flanking region of the gene is sufficient to directcells have been reviewed recently (7) and are summa-reporter gene activity to astrocytes with high specificity. Thisrized only as needed to provide a framework for dis-segment also supports a response to brain injury by upregulationcussing the transgenic experiments.of the activity. Developmentally, the transgene activity is seen

by e12.5, several days earlier than GFAP protein or mRNA hasbeen detected. GFAP transcription control regions have alreadybeen used to express several proteins in astrocytes to evaluate

IN VITRO STUDIEStheir biological effects. These proteins include IL-3, IL-6, TGF-b1, the HIV envelop protein gp120, the MHC Class I Db protein,somatosatin, CNTF, and the herpes simplex virus thymidine ki- Genomic clones have been obtained for the humannase. In the future many other GFAP transgenes are expected to (8), mouse (9–11), and rat (12, 13) GFAP genes. Aboutbe produced, with increasing knowledge of the GFAP regulatory 2 kb of the 5*-flanking region has been sequenced forelements promising greater sophistication through promoters each (10, 11, 13–15), as well as the entire transcribedthat can be regulated, have higher activity, and target activity

region for human (M. Brenner and E. Browning, un-to particular brain regions. q 1996 Academic Press, Inc.published experiments) and mouse (9) and portions ofthat for rat (12, 13, 15). Each gene is composed of nineexons distributed over about 10 kb of DNA (see Fig. 1),and yields a mature mRNA of about 3 kb. The coding

Glial fibrillary acidic protein (GFAP) is an intermedi- sequences of the three genes are highly homologous.Strong homology also extends upstream of the RNAate filament protein found almost exclusively in ma-

ture astrocytes. Because of its specificity and abun- start site for about 200 bp, recurs between about01300and 01700 (RNA startpoint Å /1) (6, 13, 14), and isdance, it has become the most commonly used marker

for astrocytes in both clinical and basic studies (represent in some intronic regions (7). The primary sitesfor the initiation of RNA and protein synthesis are es-viewed in 1, 2). Synthesis of the protein is activated as

astrocytes mature and is significantly increased as part sentially identical for each of the three genes, and eachcontains a TATA-like sequence (CATAAA or AATAAA)of the reactive gliosis that occurs in response to most

brain injuries (reviewed in 2, 3). These changes in pro- in the expected 5*-flanking position. Recently, two addi-

3511046-2023/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

AID Methods 323Y / 6709$$$261 09-24-96 14:34:38 metha AP: Methods

352 BRENNER AND MESSING

tional mRNAs that start at different sites have been bution from the upstream segment, but one possibilityis a reporter gene-specific effect. Initially, when usingidentified, GFAP-b and GFAP-g. GFAP-b mRNA ap-

pears to be identical to the primary transcript (mRNA- a luciferase reporter gene like that employed by theSueoka laboratory, the Mikoshiba/Ikenaka group alsoa), except that it is slightly larger, initiating around

bp 0170 (20). It has been reported to be the major found this region to have little effect, but more recentlythey observed a strong enhancer activity when using aform of GFAP mRNA in a Schwannoma cell line and

to constitute about 5% of the GFAP mRNA in the CNS lacZ reporter gene (K. Ikenaka, personal communica-tion).(21). GFAP-g also retains the same 3 * end as GFAP-

a, but is smaller, initiating near the end of intron 1 A more marked discrepancy among the groups is inthe location of the control elements responsible for lim-(22). It has been detected in human and mouse brain

and also in mouse bone marrow and spleen. In each iting transcription to astrocytes. Several of the labora-tories observed that the regions that activate transcrip-case its abundance is at least 10-fold lower than that

of GFAP-a in the brain. The different tissue distribution (solid bars in Fig. 1) also control its cell specificity(open bars), whereas others concluded that these func-tions of the GFAP-a, -b, and -g mRNAs suggests that

the synthesis of each is subject to unique controls. All tions are served by distinct regions. In particular, ele-ments downstream of the RNA start site were foundtranscriptional studies to date either have explicitly

measured GFAP-a or have not distinguished among necessary for appropriate specificity by the Sueoka andCowan laboratories, but not by the other three. Thethe possible mRNA isotypes.

Five groups have identified regulatory elements reason for these differences is unclear.Another unresolved issue is the importance of thewithin the GFAP genomic clones on the basis of results

from cell transfection experiments (reviewed in 7). As region immediately downstream of the RNA startpoint.Using cell-free extracts, Nakatani et al. (23) showedshown in Fig. 1, there are both agreements and discrep-

ancies among the results. Each group has found that that the region between bp /10 and /40 was criticalfor basal transcriptional activity and then supportedthe approximately 200-bp region immediately up-

stream of the RNA start point is critical for activity. this conclusion with experiments in transfected cells(24). However, Hatch and Sarid (17) found no effect ofAlso, four of the laboratories have found that a several

hundred-bp region farther upstream is important. Sig- this region on activity in cell transfection experimentsand presented evidence that the results previously re-nificantly, both of these regions fall within the homolo-

gous segments noted above. It is not known why the ported for transfected cells (but not from in vitro tran-scription) may have been due to a translational effect.Sueoka laboratory did not observe a significant contri-

FIG. 1. Summary of GFAP transcription analyses in transfected cells. The extended bars represent GFAP genomic DNA, with the positionsof the nine exons shown by solid black; the exons are numbered at the bottom. The black rectangles above each DNA segment show thepositions of elements found by the indicated laboratory to be responsible for activating transcription; the open rectangles below each segmentshow the positions of elements found to control the astrocyte specificity of expression. For details see the text and (7).


353GFAP TRANSGENIC MICE

Definitive resolution of this issue is of importance be- This latter procedure for producing transgenic miceis much faster and technically simpler than homolo-cause it influences where the junction between the

GFAP promoter and a reporter gene should be posi- gous recombination, but has the disadvantage thatboth the pattern and the strength of expression of thetioned. As long as a possibility remains that this down-

stream region contributes to gene activity, it is proba- transgene can be affected by the site of integration.This variability dictates that multiple independentlybly prudent to include it in any GFAP promoter to be

used in transgenic mice. Since this region includes the produced animals be analyzed to obtain a credible con-clusion. Several laboratories have reported that specialprotein start site at /15, care must be taken to create

in-frame protein–protein fusions or to remove tran- DNA ‘‘insulator’’ sequences can be attached to the endsof a transgene to render its activity integration-site-scribed GFAP ATGs that precede the reporter gene.independent (28–30). Perhaps because of the addi-tional effort required to generate such constructs, useof insulators has not yet been widely adopted.

TRANSGENES AND TRANSGENIC MICE In our laboratory we have found it convenient toassay for the presence of the transgene by performingpolymerase chain reactions (PCR) on DNA isolatedTransgenic mice provide a means to test the action

of genes in all cell types, developmental stages, and from a tail segment snipped off after weaning. Otherlaboratories routinely use Southern analyses or DNAphysiological states (reviewed in 25). Many transgenes

are chimeras composed of a transcriptional control re- dot blots. These latter two methods can also yield thetransgene copy number, with the Southern analysesgion attached to a heterologous sequence that is to be

expressed. In this review we will refer to the former as further confirming the global intactness of the trans-gene. Although the copy number is often measured,the ‘‘promoter’’ and the latter as the ‘‘expressed gene’’

or ‘‘structural gene.’’ In some experiments the element generally no correlation is observed with expressionlevel. This lack of concordance is attributed to aof interest is the promoter, with analysis being carried

out to test its regulatory repetoire. The structural gene stronger influence of the integration site on activity. Ithas been reported that expression does become copyto which it is linked is then termed a reporter gene, as

its function is to produce a product whose level reports number dependent if insulator segments are used (30).The time required to produce random insert trans-the activity of the promoter. In other experiments it is

the biological function of the structural gene that is of genic mice is governed by the gestation period, whichis about 20 days. To establish lines from a founderinterest. In most of these latter cases, a promoter that

restricts activity to a specific tissue or developmental requires at least another 2.5 months; the founder mustreach sexual maturity, a new generation must be born,stage is selected so that interpretation of the results is

simplified. For both cases it is thus generally critical and the offspring must be analyzed for transgene trans-mission. This additional time, as well as the resourcesthat the structural gene does not contain sequences

that influence the pattern of expression. Genes used as required for breeding, can be saved by analyzingfounder mice rather than their offspring. However,reporters should in addition encode a product that is

easy to assay, that is readily distinguished from any greater variability can be expected when analyzingfounders as they may be chimeric for the transgene orrelated endogenous entity, and that has no biological

effect on the tissues in which it is expressed. carry inserts at multiple sites. Accordingly, it is advis-able to analyze more animals than one would if workingAlthough the use of transgenic mice allows testing

of genes in a more physiological and universal setting with their offspring. The more conservative approachis to analyze established lines, as results may be veri-than does transfection of cultured cells, it is also more

demanding of time and resources. Two types of fied with multiple animals carrying the transgene inthe same integration site, and the lines may remaintransgenic mice are currently popular. In one type the

endogenous gene is specifically modified by homologous available for subsequent studies. In most cases, alltransgenic animals of a given line display very similarrecombination (26). This provides the most rigorous

testing of transgenes as they remain as close as possi- phenotypic characteristics. However, in some instanceswide variations are observed. For example, as will beble to their native state. However, as it can take over

a year of expert effort to generate a mouse of this type, described later, Galbreath et al. (31) obtained a line ofmice carrying a GFAP-TGF-b1 transgene in whichit is not yet practical for testing of multiple constructs

and so has found favor primarily for gene knockout most animals die young of a severe hydrocephalus, buta few are essentially phenotypically normal. The rea-studies. The more accessible transgenic mouse is pro-

duced by the random incorporation into chromosomes son for such incomplete penetrance of transgene activ-ity is not understood.of recombinant DNA fragments that are injected into

the pronuclei of fertilized eggs (see 27 for methodologi- Construction of transgenes is an uncertain sci-ence, but there are some general guidelines. Whencal details).



the promoter includes the RNA start site, as is the and replaced with another coding sequence, andcase for GFAP, any ATG in its transcribed portion other unique restriction sites are available for excis-should be removed by site-directed mutagenesis to ing the transgene from the vector sequences, whichprevent suppression of protein synthesis from the might otherwise interfere with proper transgene ex-structural gene. An alternative is to form an in- pression.frame fusion between the promoter and the gene to The lacZ sequence present in the placF plasmid isbe expressed. This has worked for GFAP– lacZ con- the most commonly used reporter gene for monitor-structs (32), but in other cases the chimeric protein ing the activity of transcriptional control elementsproduced could vary from the native product in stain transgenic mice. The primary attraction of thebility, localization, or activity. lacZ gene is the ease with which the encoded b-galac-

Current wisdom favors the use of genomic clones tosidase protein may be detected by histochemicalrather than cDNA for the structural gene. This is staining. However, endogenous b-galactosidase ac-based on the finding that introns are required for tivity can make detection of low levels of transgeneefficient expression of at least some transgenes (33) activity difficult, especially in kidney. A more spe-and observations, more anecdotal than published cific solution assay (43) or immunological detection(34), that introns endogenous to the expressed gene may be more discriminating. Another alternative isare more efficacious than heterologous ones. On the to measure the levels of lacZ mRNA, although forother hand, potential drawbacks of using a genomic reasons that have not been established, it is oftenclone are that it may substantially increase the size difficult to detect (M. Brenner, unpublished experi-and complexity of the construct, that its introns may ments; R. D. Palmiter, personal communication). Acontain regions that alter the transcription pattern recently described PCR protocol may prove useful(35, 36), and that it may simply not be available. The when it is necessary to measure the mRNA level,resources and requirements of a particular experi- although the possible production of aberrant tran-ment will therefore incline one toward use of a geno- scripts may be a problem (44).mic or cDNA version of the expressed gene. Several The lacZ reporter gene has now been used in hun-cDNAs have been successfully expressed using dreds of transgenes without evidence that its expres-GFAP promoters (see Biological Testing of GFAP sion is deleterious. The only suggestion we have seenTransgenes, below). Other considerations for the ex- of a possible pathological effect is the observation ofpressed gene are the lengths of the untranslated re- M. Galou et al. (45) that a nuclear-targeted lacZ maygions (UTRs) to be included and the context of the cause an alteration in chromatin structure. Otherprotein initiation site. It may be advisable to limit

reporter genes that have found favor include chlor-the length of the UTRs as the 5* UTR is a commonamphenicol acetyltransferase (constructs are oftenlocation for transcriptional control elements (37),already in hand from cell transfection experiments),and the 3 * UTR for sequences that destabilize mRNAluciferase (its assay is extremely sensitive), and b-(38). A very short 5* UTR may hinder translationglobin (its relatively stable mRNA facilitates its de-(39), but sufficient length is probably contributed bytection). Green fluorescent protein (46) is a relativelythe GFAP promoters to be described later that a con-new reporter gene with the potential for in vivo iden-tribution from the structural gene is not needed. Iftification of expressing cells. While it is rarely ob-the context of the protein initiation site suggestsserved that these reporter genes influence the pat-that it is weak, it may be worthwhile to substitutetern of expression, this can occur (47, 48). Thus inthe consensus sequence reported by Kozak (40).some instances it may be necessary to test promotersWhen a cDNA construct is used for a transgene, awith more than one reporter gene. In the case of thepreviously tested heterologous intron and polyade-GFAP promoter, assays of the expression of othernylation site can be supplied as part of the cloningstructural genes have largely confirmed the resultsvector. The SV40 small t intron, present in some vec-obtained with lacZ (see Biological Testing of GFAPtors, should be avoided, however, due to the possibil-Transgenes, below).ity of aberrant splicing (41). We have had good suc-

A long-sought goal that should greatly expand thecess using the lacZ-containing vectors created by J.utility of transgenic analyses may have been met withPeschon in the laboratory of R. D. Palmiter (42).the recent description of transgenes subject to tightThese plasmids contain a multicloning site for inser-regulation by tetracycline (49, 50). If the preliminarytion of a promoter upstream of a cytoplasmic (placF)results hold up, it will be possible to dramatically alteror nuclear-targeted (pnlacF) Escherichia coli lacZthe level of transgene expression by manipulating tet-gene. A segment of the mouse protamine-1 gene (mP-racycline levels in the animal. This would allow the1) follows to supply an intron, a 3 * UTR believed tostudy of transgenes whose continuous activity is lethalconfer mRNA stability, and a polyadenylation site.

BamHI sites flank the lacZ gene so it can be excised or produces confounding effects.



transgene was given the designation GFAP1 to distin-ANALYSIS OF GFAP PROMOTER ELEMENTS guish it from a modified version, GFAP2 (52, 53), thatIN TRANSGENIC MICE was constructed to incorporate several of the design

features discussed under Transgenes and TransgenicMice above. In GFAP2 the insertion site for the ex-General Commentspressed gene was moved further 3 * to bp /92 to ensure

Studies of the GFAP promoter have been undertaken retention of the complete basal promoter element de-in transgenic mice to determine if deductions made scribed by Nakatani et al. (23), and the two transcribedfrom in vitro experiments are valid for the living ani- ATGs prior to this site were converted to TTGs so thatmal. Such verification is important as it is not unusual protein synthesis would commence within the ex-to find that the expression of genes in cultured cells is pressed gene rather than creating a protein–proteindifferent from that in the animal (35). This likely occurs fusion. In addition, an intron from SV40 was placedbecause the physiological state of cells in culture does immediately before the expressed gene, and as withnot accurately reproduce that in vivo. In addition, it is GFAP1, the expressed gene was followed by an SV40not now, and may never be, possible to replicate and polyadenylation signal (see Fig. 2). A detailed compari-test in culture the combinatorial totality of cell types, son of the activities of the GFAP1 and GFAP2 promot-developmental stages, and physiological states that oc- ers has not been reported, but those that have beencur in an animal as complex as the mouse. made indicate that they have very similar activities

Analysis of GFAP transcription in transgenic mice (52, 54).has occurred in two stages. Experiments were first con-ducted to determine if a genomic segment of the gene 2-kb Promoterscould mimic the effects of the endogenous gene. This Very similar results were obtained by our group us-has been followed by testing subfragments to more spe- ing a human promoter corresponding closely to the 5*cifically identify the control regions. segment of the above construct (43). Our 2.2-kb pro-

moter, termed gfa2, commences at bp 02163 and is13-kb Promoters joined at bp /47 to the lacZ gene. The normal protein-

initiating ATG at bp /15 was changed to TTG by site-The first report of a GFAP transgene came from theMucke laboratory (51). They tested a 13-kb mouse directed mutagenesis so that synthesis of the reporter

protein would commence with the lacZ gene. Two ver-GFAP genomic fragment that commenced about 2 kbupstream of the RNA start site, extended through the sions of the lacZ reporter were used, one encoding the

standard cytoplasmic form of the enzyme and anotherentire 10-kb transcription unit, and terminated about1.5 kb beyond the polyadenylation signal. Activity was incorporating the SV40 large T antigen nuclear local-

ization signal to target the enzyme to the nucleus. Themonitored by placing the E. coli lacZ gene within thefirst exon at bp /43, such that its coding region was cytoplasmic version is useful for determining the mor-

phology of expressing cells, whereas the nuclear ver-in-frame with the GFAP protein start at /15 (seeGFAP1 in Fig. 2). The lacZ gene was followed by an sion is better suited for counting cells and double stain-

ing using immunoperoxidase methods. As describedSV40 polyadenylation signal to prevent the down-stream GFAP sequences from being included in the under Transgenes and Transgenic Mice above, both re-

porter gene cassettes conclude with a segment of thetransgenic mRNA. Using histochemical staining andNorthern analyses, lacZ activity was detected in the mouse protamine-1 gene to provide an intron, stabiliz-

ing 3 * UTR, and polyadenylation signal. We have foundbrain of transgenic animals, but no activity over theendogenous level was observed in liver, spleen, heart, no differences in the level or specificity of expression

of the nuclear and cytoplasmic versions.lung, sciatic nerve, kidney, salivary gland, thymus, tes-tis, epidydimis, or pancreas. Activity in the brain was Using a fluorimetric solution assay for b-galactosi-

dase, the lacZ reporter gene was found to expressconfined to astrocytes based on its spatial distribution,the morphology of the expressing cells as seen by light strongly in the brain (up to 80 times background), but

not in the kidney, liver, heart, lung, or spleen. The cellmicroscopy, and double staining for GFAP.Importantly, following a stab wound local expression specificity of brain expression was evaluated in two

ways. In one procedure, sections of the optic nerve wereof the transgene was rapidly induced, within 1 h, indi-cating that the transgene contained elements directly stained for b-galactosidase with Bluo-gal, which pro-

duces an electron-dense product, to permit ultrastruc-responsible for the elevation of GFAP transcription byinjury-induced mediators. The stimulated activity of tural identification of expressing cells. No neurons or

oligodendrocytes expressed the transgene, whereasthe transgene reached a peak and then decayed morerapidly than that of the endogenous gene, perhaps due most, but not all, of the astrocytes were stained. In

the second method, cultures of differentiated cells wereto differences in mRNA stabilities.Recently the promoter of this original GFAP– lacZ obtained from EGF-responsive neural stem cells (55)



prepared from transgenic animals. Double staining for two- to fourfold increase in GFAP (58). During embry-onic development, activity of the gfa2–lacZ transgenelacZ activity and cell type-specific antigens (neurofila-

ment, proteolipid protein, and GFAP for neurons, oligo- could be detected histochemically as early as e12.5,whereas the endogenous GFAP gene activity had notdendrocytes, and astrocytes, respectively) again re-

vealed expression to be confined to astrocytes (J. been seen until e16.5 (59, 60). In four of the five linesexamined, this early activity was confined to presump-Hammang, unpublished experiments). Like the larger

promoter, the gfa2–lacZ transgene responded to brain tive astrocyte precursors in the dorsal spinal cord andin the ventricular zone of the telencephalon and hippo-injury; a local increase in b-galactosidase production

was observed when assayed 2 days following a stab campus. In the fifth line signal activity also appearedtransiently in chondrocytes.wound to the cerebral hemisphere.

Quantitative assays of gfa2–lacZ transgene activity We have offered three possible explanations for thetiming difference between the transgene and endoge-in the brain following birth have shown that the level

increases for 1 to 2 weeks and then plateaus or falls nous GFAP activities (43): it may reflect a differencebetween human and mouse regulation; regulatory se-slightly. These kinetics are similar to those observed

for endogenous GFAP (56, 57). Transgene activity has quences required for silencing the gene at early stagesmay be missing from the transgene promoters; or thenot yet been monitored in old animals, which show a

FIG. 2. Structures of GFAP promoters and lacZ reporter genes. Diagrams of several of the GFAP promoters discussed in the text arepresented along with the structures of the lacZ reporter genes to which each was linked to evaluate its activity. The empty brackets indicatethe insertion site for a gene to be expressed (e.g., lacZ). Abbreviations not already defined in the text are as follows: SV40 polyA, polyadenyla-tion signal of the SV40 late region; SV40 intron, SV40 late region intron; SV40 nls, SV40 large T nuclear localization signal. For the reportergene used with the gfa2 promoter, an intron and polyadenylation signal are provided by a fragment of the mouse protamine-1 gene (mP-1). For the nuclear-targeted version of the gfa2–lacZ transgene, the lacZ coding sequence was preceded by the SV40 nls. The positions ofthe exons following the expressed gene insertion site are indicated by black rectangles. The figure is not drawn to scale.



endogenous gene may indeed be on at the same time, human GFAP as the transgene. The mouse and humanbut its activity may not be as easily detected as that transcripts are remarkably similar (7), but can beof b-galactosidase. The first possibility has now been readily distinguished by probe protection assays (M.eliminated, as H-Y. Chung, L. Mucke, and A. Messing Brenner, unpublished experiments).(unpublished experiments) have recently found that While our gfa2–lacZ and each of Mucke’s GFAP–the mouse GFAP2– lacZ transgene described above is lacZ transgenes gave very similar results, there wereexpressed with a developmental profile similar to that some provocative differences in the topography of theirof the human gfa2–lacZ transgenes. This result also expression. The gfa2–lacZ transgene was expressedimpacts on the second possibility, since any putative fairly uniformly in astrocytes throughout the brain, in-silencer element must now lie outside the 13-kb geno- cluding Bergmann glia, whereas expression of both themic fragment, which extends 3 * to about 1.5 kb beyond larger (GFAP1 and GFAP2) and smaller (GFAP3)the polyadenylation signal. In addition, H-Y. Chung GFAP– lacZ transgenes from the Mucke laboratoryand A. Messing (unpublished experiments) have ob- was seen predominantly in the hippocampus, dentateserved a similar developmental profile when the pro- gyrus, and white matter tracts and was not readilymoter of the human GFAP– lacZ transgene just de- detected in Bergmann glia. In addition, the Muckescribed is extended upstream to 5 kb 5* of the RNA transgenes are expressed in lens epithelium and satel-start site, further increasing the exclusion zone for a lite cells of the dorsal root ganglia, but not in spinalsilencer. On the other hand, the third alternative, that cord astrocytes, whereas the reverse is found for gfa2–the difference is an artifact of detection sensitivity, has lacZ (32, 43, 54, H.-Y. Chung, L. Mucke, and A. Mess-received support from the earlier detection of GFAP ing, unpublished experiments). Although it is seductivemRNA than previously reported. Using Northern anal- to attribute these differences in expression to speciesyses, B. Teter et al. (61) observed the mRNA at e14. variation between human and mouse, it is also possible

Johnson et al. (32) have recently conducted a compar- that they are due to the other sequences present inison between the activity of their larger, GFAP2 pro- the constructs—the mP-1 segment in gfa2 or the intronmoter and a smaller one comparable to the human gfa2 or polyadenylation-containing segment of SV40 inpromoter. Their smaller promoter, which we will call GFAP1, -2, and -3 (Fig. 2).GFAP3, is identical to GFAP2, except that all GFAP The above results demonstrate that the 2-kb GFAPsequences following the expressed gene, i.e., after

promoters are capable of directing expression with highGFAP bp /92, have been deleted (Fig. 2). There wasspecificity to astrocytes, a conclusion that has been sup-a suggestion that the smaller transgene may expressported by their use in several applied studies (see Bio-somewhat more often in nontarget tissues than thelogical Testing of GFAP Transgenes, below). However,larger version, but insufficient numbers of lines wereusing quite similar promoters, two other groups havegenerated to establish this point. In all other respectsfound significant ectopic expression. Schonrich et al.the behaviors of the two transgenes were essentially(62) linked a mouse GFAP fragment that extended fromindistinguishable.about 02700 to /92 to a genomic clone of the MHCOf particular importance, Johnson et al. (32) carriedclass I Kb gene. In addition to expression in astrocytes,out a quantitative comparison between the two trans-the Kb protein was detected by immunohistochemistrygenes for basal and reactive activities, finding that bothin ependymal cells, some choroid plexus epithelial cells,produced similar levels of expression. Thus the 11.5-enteric glia, and cultured oligodendrocytes. A more de-kb region extending from /93 to about 1.5 kb down-tailed study was carried out by M. Galou et al. (45),stream of the GFAP polyadenylation signal apparentlywho joined a fragment extending from 01913 to /92does not contain any critical qualitative or quantitativeof the Mus spretus GFAP gene to lacZ (Mus musculusregulatory regions, with the caveat that a contributionwas used by the other groups). Their promoter, whichcould have been compromised by its being displacedwe will call GFAP (spretus), produced expression inabout 3 kb farther 3 * by insertion of the lacZ gene.astrocytes in both gray and white matter, with greatestIt remains to be determined if the level of activity ofactivity appearing in the corpus callosum, septum, cer-the GFAP promoters used is comparable to that of theebellar white matter, and spinal cord. Although it wasendogenous gene. We have obtained a rough estimatesignificantly lower than that in astrocytes, activity wasof this by using probe protection assays to compare thealso seen in several populations of neurons, includinglevels of the endogenous GFAP mRNA to that of thecerebellar granule cells and neurons within brain corti-herpes simplex virus thymidine kinase (HSV-TK)cal layers and the hippocampus. No staining of oligo-driven by the gfa2 promoter (see below). Similar levelsdendrocytes was observed. During development lacZwere found. However, this estimate could be in erroractivity was seen in cerebellar neurons by e14 and inif the two RNA transcripts differ substantially in theirastrocytes by e18. Transient ectopic expression wasprocessing or stability. To obtain a more dependable

result, we have experiments underway using genomic also reported in tongue, thoracic duct, and thymus.



Stab wounds to the brain increased local activity of the that following induction of reactive gliosis in the mouseeye, GFAP protein persists at elevated levels for sev-transgene.

There is no clear explanation of why these latter two eral weeks after GFAP mRNA levels have declined.Thus it is possible that the transcriptional activities ofstudies differ from each other and from those of our

laboratory and that of Mucke. In the case of Schonrich the endogenous and transgenic promoters are actuallyquite similar. Whether or not this is the case shouldet al. (62), only one line of mice was examined, so an

integration site artifact is possible. It is also possible be resolved by analysis of the mice carrying the humanGFAP transgene.that the introns of the Kb gene affected the tissue pat-

tern of expression. In addition, the two ATGs in theSubregions of the 2-kb PromoterGFAP 5* UTR were left intact, and it is unclear whether

the linkage with the Kb gene produced a protein–pro- Cell transfection studies identified two variants ofthe gfa2 promoter that were of particular interesttein fusion.

The procedures of Galou et al. (45), on the other hand, (14). One, called gfa28, is a much smaller versioncomposed of an upstream regulatory region (bpclosely parallel those of our group and Mucke’s. All

three laboratories analyzed multiple independent 01757 to 01489) joined directly to a promoter proxi-mal regulatory region (0132 to 057) and basal pro-lines, ruling out integration site artifacts. All three also

analyzed their transgenes in the same genetic back- moter (056 to /47). The other, gfa57, has the GFAPbasal promoter (056 to /47) replaced with the SV40ground of C57BL/6 1 SJL/J hybrids (identical results

with gfa2–lacZ transgenes have also been found in an early gene basal promoter. Limited cell transfectiondata had suggested that both constructs would re-FVB/N background (43)). The close agreement between

our results using a human GFAP promoter and tain astrocyte specificity of expression, while aug-menting activity 10-fold over the gfa2 level.Mucke’s using a M. musculus promoter suggests that

the presumably smaller differences that may exist be- These promoters are of interest for further localiz-ing the critical regulatory regions of the GFAP genetween M. musculus and M. spretus are unlikely to be

a factor. Other design features of the constructs also and for the potential practical purpose of having asmaller and/or more potent astrocyte-specific ex-appear to be very similar (Fig. 2). Like GFAP1– lacZ,

GFAP (spretus)– lacZ contains an in-frame fusion be- pression cassette. Each has now been linked to thelacZ reporter gene, and some preliminary resultstween GFAP and lacZ, and the lacZ gene is followed by

an SV40 polyadenylation signal; and like our nuclear- have been obtained in transgenic mice. Solutionassays of various organs for b-galactosidase suggesttargeted gfa2–lacZ, the lacZ gene of GFAP (spretus) is

preceded by the T antigen nuclear localization se- that the tissue specificities of gfa28 and gfa57 areidentical to that of gfa2. The gfa28– lacZ transgenequence. The only feature of note unique to the Galou

et al. construct is the absence of any intron. Whether (but not gfa57– lacZ) has also been tested for cellspecificity using the neural stem cell procedure andthis or some other subtle variation in procedure is re-

sponsible for the different patterns of activity observed found to express only in astrocytes (J. Hammang,unpublished experiments). In addition, it respondsremains to be determined. One feature that all of the

promoters do have in common is that a significant por- with elevated activity to a stab wound (A. Messing,unpublished experiments). Unexpectedly, however,tion of GFAP-containing astrocytes in the brain fail to

stain for b-galactosidase. Part of this expression defi- both gfa28–lacZ and gfa57– lacZ show activity dis-tribution patterns in the brain that differ from theciency is regional in nature, with activity of some of

the lacZ transgenes being favored in particular brain uniformity found for gfa2. In the adult, gfa28 is ex-pressed almost exclusively in the cortex, hippocam-domains. Results presented below for subregions of

GFAP promoters raise the possibility that regional ac- pus, and caudal vermis of the cerebellum, whereasgfa57 is expressed in all brain regions, but mosttivity may be controlled by the presence or absence

of particular transcriptional elements. However, in all strongly in the cerebellum. Preliminary quantitativedata suggest that contrary to the cell transfectioncases GFAP-containing b-galactosidase-positive astro-

cytes were seen as well in the immediate vicinity of results, in those brain regions in which gfa28 is ac-tive, it does not express at a markedly higher levelmorphologically identical b-galactosidase-negative as-

trocytes. Either the heterogeneity among astrocytes is than gfa2. On the other hand, initial results suggestthat the activity of gfa57 is significantly elevatedfar more subtle and ubiquitous than previously consid-

ered or these differences reflect a stochastic technical over that of gfa2. Developmental expression ofgfa28–lacZ has also been monitored. Activity is de-artifact. For example, activation of the GFAP promoter

may be episodic. Since GFAP is a fairly stable struc- tectable by e12.5, like for gfa2–lacZ, but the rangeof expression is more limited throughout embryogen-tural protein, it may persist between intervals of gene

activity, whereas b-galactosidase protein may be de- esis, consistent with the spatially restricted patternseen in the adult.graded. For example, Sarthy and Egal (63) observed



The results obtained with gfa57 indicate that the ments are present in the transgene but are generallyrepressed by some feature of transgene integration.GFAP basal promoter is not required for tissue speci-

ficity (cell specificity within the brain for this pro-moter has not been examined). Combining this ob-servation with those for gfa28 suggests that the BIOLOGICAL TESTING OF GFAP TRANSGENEScritical determinants for gene activity lie within the01757 to 01489 and 0132 to 057 regions. These

Considerations and Caveatsregions also contain at least one gliosis response ele-ment responsible for increasing gene activity after The promise of directly expression of genes to astro-

cytes was a major motivator of the above studies. Theinjury. On the other hand, the limited distributionof gfa28 activity means that there are regulatory ele- results described indicate that several GFAP promot-

ers that should serve this purpose well are now avail-ments lying outside of these segments that are es-sential for expression in certain brain regions, like able, and indeed a number of successful applications

have already been realized and are summarized below.the thalamus, brain stem, most of the cerebellum,retina, and spinal cord. A corollary is that astrocytes Which of the several promoters is best suited for con-

structing a transgene will depend on the particular re-in different brain regions use different sets of regula-tory elements for controlling GFAP transcription. As quirements of the investigation. The 2-kb promoters,

gfa2 and GFAP3, are technically easier to work with,a practical note, it may be possible to exploit thesedifferences to create a family of brain region-specific but the suggestion that the 13-kb promoters GFAP1

and GFAP2 are less likely to display ectopic expressionexpression cassettes.may incline one toward their use. Between gfa2 andGFAP3, the choice is clearly the former if expressionActivity in Nonastrocytic Cells That Express GFAPin Bergmann glia or spinal cord astrocytes is required,

Although GFAP is expressed predominantly in as- but GFAP3 for expression in lens epithelium. If prelim-trocytes, it is also found in several other cell types, inary results hold up, gfa57 may be favored when theincluding Bergmann glia, lens epithelium, reactive strongest available activity is needed. As studies con-retinal Muller cells, nonmyelinating Schwann cells, tinue, researchers can look forward to GFAP promoterenteric glia, liver perisinusoidal stellate cells (all re- cassettes that can be regulated, are more active, andviewed in 1), and testicular Leydig cells (64). Expres- are specific for particular brain regions.sion in Bergmann glia is seen in both the human Whichever promoter is selected, consideration mustgfa2 promoter (43) and the mouse GFAP1, -2, and be given to the possibility that the expressed gene could-3 promoters; for these mouse promoters expression alter the specificity that was documented using lacZ;was difficult to discern with the lacZ reporter gene thus some degree of confirmation of the expression pat-(32), but was readily apparent with IL-6 (52). Ex- tern is requisite for each new transgene used. A changepression in lens epithelial cells was also seen with in the expression pattern could result either from athe mouse promoters (32, 54), but not with gfa2 (A. primary effect on transcription or through a chain ofMessing, unpublished experiments). With one excep- indirect causes. An example of a primary effect is thetion, neither the human (43, A. Messing and M. finding that sequences within the HSV-TK gene acti-Brenner, unpublished experiments) nor the mouse vate its expression in testes, rendering transgene-ex-promoters (32, 51, 54) gave detectable expression in pressing males sterile (65, 66). We have noted this out-reactive Muller cells, Schwann cells, enteric glia, come in gfa2–HSV-TK mice that we are currentlyperisinusoidal stellate cells, or Leydig cells. For analyzing (see below). A common indirect effect forsome of these cell types, analyses were at the whole GFAP-driven transgenes is induction of reactive gliosistissue level, so localized expression by the minor cell by the expressed gene product, resulting in a positivepopulations believed to express GFAP could have feedback loop for transgene expression. This has beenbeen overlooked; however, a careful search was made observed for the expression of TGF-b1 (31, 67), IL-6for possible expression in Schwann cells and reactive (52), and gp120 (53) transgenes. In addition to the re-Muller cells for both promoter types and also in en- sultant quantitative increase in transgene expression,teric glia and perisinusoidal stellate cells for gfa2– the GFAP1-IL-6 transgene also displayed a differentlacZ. The exception is that one of the six lines of topographical distribution from that described forgfa2– lacZ transgenics examined, line TgNMes3, GFAP1– lacZ; in particular, it was strongly expresseddoes express in nonmyelinating Schwann cells and in Bergmann glia. This may also be due to a secondaryenteric glia (43, A. Messing, unpublished experi- effect on transcription, as when a GFAP1– lacZments). It is possible that this appropriate expres- transgene was co-incorporated with a GFAP1–IL-6sion is a fortuitous artifact of its integration site or transgene, lacZ expression was similar to that of IL-6

(52). One explanation is that certain classes of astro-it could suggest that the required regulatory ele-



cytes are more prone to a reactive response to IL-6, Class I Kb gene and the gp120 protein have been pre-viously reviewed (7); here we focus on results reportedleading to differential activation of the expressed gene.

It is thus possible that the potential positive feedback during the past 2 years, including some data that haveappeared only in abstract form.loop between reactive gliosis and expression of a GFAP

The simplest application of GFAP-driven transgenestransgene may sometimes complicate analyses; how-is to use the lacZ animals already described to markever, in other cases this feature can be exploited toastrocytes in transplantation studies. Mucke employedupregulate the gene to be expressed at the precise timethis approach to follow the fate of transgenic astrocytesthat its biological function is of interest.injected into a nontransgenic host (79), and we are cur-

Applications rently using it to facilitate identification and analysisAll studies we know of that have used GFAP-expres- of astrocytes in GFAP-null mice.

sion cassettes to assess the function of an expressed A number of studies have used GFAP transgenicmice to address the mechanisms of CNS inflammatorygene are listed in Table 1. Those involving the MHC

TABLE 1

Results Obtained with GFAP Transgenes

CategoryPromotera Structural geneb Results References

AutoimmunityMouse 2.7 kbc MHC class I Kb gene Tolerance induced by extrathymic expression of antigen 62

b2 microglobulin geneMouse GFAP1 MHC class I Db minigened Astrocytes could act as antigen-presenting cells in cell culture 68

CTL assay

CytokinesMouse GFAP2 IL-6 cDNA Neurodegeneration, astrocytosis, neovascularization, microglial 52, 69–71

activation, failure of blood–brain barrier to form, andphysiological alterations in hippocampal neurons

Human gfa2 TGF-b1 cDNA Hydrocephalus, beginning in early postnatal period 31Mouse GFAP2 TGF-b1 cDNA Hydrocephalus, associated with increased expression of 67

laminin and fibronectinMouse GFAP2 IL-3 cDNA Diffuse microglial activation, meningoencephalitis, astrocytosis, 72, 73

neurodegeneration, and primary demyelinationMouse GFAP2 Interferon a1 cDNA Progressive vascular mineralization and neurodegeneration 74

Viral diseaseMouse GFAP2 HIV gp120 (aa1–509) Vacuolization of neuronal dendrites, loss of large pyramidal 53

cDNA neurons in neocortex, diffuse astrocytosis

Cell ablationHuman gfa2 HSV-TK genee Treatment with ganciclovir during first postnatal week caused 75, 76

disordered radial glia, loss of astrocytes, depletion of granulecells, ectopic Purkinje cells, and stunted Purkinje celldendrites

Growth factorsMouse 2.0 kb f CNTF cDNA Increased motoneuron survival, during development (CNTF-ns) 77

or following axotomy (CNTF-s)Human gfa2 Somatostatin gene Hyperactivity, primarily in females 78

MiscellaneousMouse GFAP2 a1-antichymotrypsin cDNA No apparent pathologyg

a Unless otherwise indicated, structural details of these promoters are given in the text; see Fig. 2 for illustrations of the mouse GFAP1,mouse GFAP2, and human gfa2 promoters.

b Unless otherwise indicated, ‘‘gene’’ refers to clones containing endogenous introns, and ‘‘cDNA’’ to cDNA fragments combined withheterologous introns.

c This promoter extends from about bp 02600 to /92.d The minigene contains only the first and second introns of the Db gene.e Two forms of this transgene were used, one with an heterologous intron derived from the mouse protamine-1 gene as illustrated for the

human gfa2 promoter in Fig. 2, and one without introns and using the endogenous HSV-TK polyadenylation signal.f This promoter extends from bp 01995 to /7 (J. T. Henderson, personal communication).g L. Mucke, personal communication.



disease and autoimmunity. In one example, Mucke’s develop an acute, severe meningoencephalitis, low-ex-pressing lines have an adult-onset, progressive multifo-laboratory tested the ability of astrocytes to act as anti-

gen-presenting cells in the CNS, by targeting expres- cal disease, especially involving the white matter of thecerebellum and brain stem, showing both demyelin-sion of the Db class I MHC gene (68). FACS analysis

of cultured astrocytes demonstrated cell specificity of ation and remyelination. The demyelination appearedto be mediated by activated macrophages and microg-expression and also that the MHC molecule was dis-

played on the cell surface. Cultures were prepared from lia. The earliest change noted was increased expressionof the MHC class II Ia gene in perivascular microglia,the transgenic astrocytes, infected with the lympho-

cytic choriomeningitis virus, and then tested in a stan- a cell type that has previously been implicated as play-ing a key role in initiating autoimmune disease in thedard CTL assay using Db-restricted splenic lympho-

cytes. These experiments demonstrated that, in vitro at CNS (85).Both the Messing and Mucke laboratories haveleast, astrocytes could present antigen to lymphocytes.

Whether they have this capacity in vivo remains to studied the effects of increasing astrocyte expressionof TGF-b1 (31, 67), a cytokine thought to regulatebe established. Since previous studies involving MHC

class I transgenes expressed in oligodendrocytes (80) astrocyte response to injury (86) and that suppressesinflammation in experimental autoimmune enceph-or pancreatic islet cells (81) both resulted in impaired

cellular function, it is interesting that expression of the alitis (87). Unexpectedly, the transgenic mice devel-oped a communicating hydrocephalus, which was fa-class I Db transgene in astrocytes by itself had little

apparent effect on the mice. A benign effect was also tal to high-expressing animals during the first fewpostnatal months. In the study of Galbreath et al.noted by Schonrich et al. (62) for expression of the class

I Kb gene in astrocytes. (31), the human gfa2 promoter was used to directexpression of a porcine TGF-b1 cDNA that had beenAnother immunological question addressed by the

Mucke laboratory is whether astrocyte antigens can mutated at critical cysteine residues to promote se-cretion of an active form of the polypeptide. All 12serve as the initial target of immune attack (82). In this

study GFAP1– lacZ transgenic mice were immunized founder mice that expressed the transgene died ofhydrocephalus between birth and weaning, but oneagainst the E. coli b-galactosidase, which would be ex-

pressed only by astrocytes. This protocol was found to breeding line was established from a moribund fe-male founder by ovarian transplantation. This lineinduce severe inflammation involving both B- and T-

cell responses, microglial activation, and secondary gli- displays variable penetrance, with some of thetransgenic mice being normal and others developingosis. To what extent bystander effects may damage oli-

godendrocytes or myelin, producing demyelination, re- hydrocephalus. The presence of the disease corre-lated with expression of the transgene. Dye injec-mains to be established. In addition, these mice show

minimal clinical deficits (83), which may reflect the tions into the lateral ventricles and serial sectionhistopathology showed that foramina and aqueductsabsence of spinal cord expression of the lacZ gene by

the mouse GFAP1 and GFAP2 expression cassettes as as far back as the fourth ventricle were all patent,establishing this as a communicating form of hydro-noted above.

Since inflammation typically involves a complex set cephalus. However, subsequent studies have shownthat the outflow tracts from the fourth ventricle toof changes in the expression of multiple cytokines, it

has been of interest to test whether expression of indi- the subarachnoid space fail to open during late ges-tation (88). A developmental effect of the TGF-b1vidual cytokines can initiate disease processes in the

CNS (for a recent review see 84). Campbell et al. (52) expression was also suggested by the presence offewer folia in the cerebellum of transgenic neonatalreported that overexpression of IL-6 in astrocytes re-

sulted in significant neuropathology, with induction of mice. TGF-b1 has previously been shown to suppressproliferation of both neuronal (89) and oligodendro-acute-phase proteins such as a1-antichymotrypsin.

Subsequent work on the same mice has shown exten- glial (90) progenitors, which may contribute to thisimpaired development.sive changes in other molecules that might augment

the immune response, including elevation of IL-1, TNF- In the study of Wyss-Coray et al. (67), the samemutated form of TGF-b1 was inserted into the firsta, and ICAM-I (70) and failure of the normal blood–

brain barrier to form during the second postnatal week exon of the mouse GFAP gene (GFAP2 expressioncassette). Five of eight founder mice died with neuro-(71). In addition, the GFAP–IL-6 mice show distinct

physiological changes in functional properties of cholin- logic symptoms between 2 weeks and 2 months post-natal. Another founder gave rise to a low-expressingergic neurons in the hippocampus (69).

Campbell’s laboratory also has generated transgenic line that was analyzed in detail. In this line, only1% of the hemizygous transgenic mice developed hy-mice carrying a GFAP2–IL-3 construct (72, 73). Like

the IL-6 mice, these animals develop significant neuro- drocephalus, but when the mice were bred to homo-zygosity for the transgene the incidence of hydro-logical disease. Whereas mice in high-expressing lines



cephalus rose to 100%. All foramina examined kill them off and see what happens. A transgene ex-pressing the HSV-TK gene under the control of a GFAPappeared patent. A distinct pattern of reactive astro-

cytosis was observed, concentrated around blood ves- promoter provides a convenient means to this end.HSV-TK expression per se is innocuous, but if animalssels, and the extracellular matrix (ECM) molecules

fibronectin and laminin were induced severalfold at are fed an anti-herpetic agent such as ganciclovir, theenzyme converts it to a toxic product that kills prolifer-both the mRNA and the protein levels. Immunohisto-

chemical detection of the ECM components showed ating cells. Single drug treatments of HSV-TK-express-ing mice during the first postnatal week dramaticallyparticular sites of increase in the meninges and

around small to medium sized blood vessels, impli- alters cerebellar development, with marked depletionof the cells in the external granule cell layer and sec-cating interference with CSF formation or its absorp-

tion in the pathogenesis of the hydrocephalus. The ondary changes in the morphology of Purkinje cell den-drites (75). A substantial loss of astrocytes in thehydrocephalus was considered not to have a develop-

mental component since no cytoarchitectural abnor- treated mice was confirmed by nuclear lacZ staining ofgfa2–lacZ/gfa2–HSV-TK double transgenic mice (76).malities were seen other than the pressure-induced

atrophy of neocortex and since it could be induced One interpretation of these results is that astrocytesare essential for production of the cerebellar granulein asymptomatic hemizygous transgenic adults by

upregulation of the GFAP-controlled transgene by cells. However, bystander killing is another explana-tion for cell loss in HSV-TK ablation experiments (94)focal injury.

Since astrocytes are thought to be important and needs to be rigorously excluded.sources of growth factor expression within the CNS,transgenic manipulation of these factors are naturalapplications of GFAP expression cassettes. Hender-son et al. (77) have forced overexpression of two CONCLUDING REMARKSforms of CNTF, either the natural nonsecreted factor(CNTF-ns) or one modified for secretion (CNTF-s).

The analyses of GFAP promoter elements inWith the CNTF-ns, motoneuron numbers (as mea-transgenic mice have shown that a 2-kb 5*-flankingsured in the facial nucleus) increased 50% over nor-segment of the gene is capable of faithfully directingmal, perhaps reflecting decreased cell death duringexpression to astrocytes and also of responding to de-development. In contrast, expression of the CNTF-svelopmental and injury-engendered cues in a mannerform did not affect developmental survival, but didsimilar to the endogenous gene. Much work remains tocause a nearly threefold increase in survival follow-determine the specific promoter sites responsible foring axotomy. In light of the adult-onset motoneuronthese activities, the critical first step for realizing thedegeneration reported in CNTF-null mice (91), itgoal of elucidating cell-signaling mechanisms throughwould be of interest to place the GFAP–CNTFidentification and analyses of the participating tran-transgene in the CNTF-null background to testscription factors. The genomic requirements for expres-whether astrocyte expression can completely rescuesion of GFAP in nonastrocytic cells also await delinea-the null phenotype. A recent report on transgeniction. However, the more practical objective of producingmice expressing CNTF under the control of its owna transcription cassette for directing the expression ofregulatory regions showed that it actually inducedgenes to astrocytes has been met. As summarized inastrocytic gliosis in certain areas of the CNS (92).this review, studies using this capability have alreadyWhether similar gliotic changes are occurring in theyielded both models of disease and insights into brainGFAP–CNTF mice is not yet clear.physiology. The number of laboratories that have re-Somatostatin, a small neuropeptide, is also thoughtcently requested and received these promoters augursto have trophic activity on certain neuronal populationsfor a short-lived currency for this review.(reviewed in 93) and to stimulate the rate of differentia-

tion of cerebellar granule cells (78). Schwartz et al. (78)analyzed transgenic mice expressing somatostatin un-der the control of the human gfa2 promoter and found a

ACKNOWLEDGMENTSstriking behavioral change consisting of hyperactivity,primarily of females. The transgenic mice displayedincreased horizontal activity and more total distance For conveying unpublished information we thank Drs. Iain Camp-

bell, Maria Galou, Joe Hammang, Jeffrey T. Henderson, Kazuhirotraveled. In older animals tested for vertical activity,Ikenaka, Lennart Mucke, Richard Palmiter, and Joan Schwartz.males rather than females were hyperactive. Anatomi-M.B. is supported by the Intramural Program of the NINDS throughcal studies of these mice are currently underway. Dr. John Hallenbeck, Chief of the Stroke Branch. A.M. is a Shaw

One method to evaluate the proposed roles for astro- Scholar of the Milwaukee Foundation and is supported by grantsfrom the National Multiple Sclerosis Society and the NIH.cytes in brain development and function is simply to



30. Neznanov, N., Thorey, I. S., Cecena, G., and Oshima, R. G. (1993)REFERENCES Mol. Cell. Biol. 13, 2214–2223.

31. Galbreath, E., Kim, S.-J., Park, K., Brenner, M., and Messing,A. (1995) J. Neuropathol. Exp. Neurol. 54, 339–349.1. McLendon, R. E., and Bigner, D. D. (1994) Brain Pathol. 4, 221–

32. Johnson, W. B., Ruppe, M. D., Rockenstein, E. M., Price, J.,228.Sarthy, V. P., Verderber, L. C., and Mucke, L. (1995) Glia 13,2. Eng, L. F., and Ghirnikar, R. S. (1994) Brain Pathol. 4, 229–174–184.237.

33. Brinster, R. L., Allen, J. M., Behringer, R. R., Gelinas, R. E.,3. Eddleston, M., and Mucke, L. (1993) Neuroscience 54, 15–36.and Palmiter, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 836–

4. Sarthy, P. V., and Fu, M. (1989) DNA 8, 437–446. 840.5. Landry, C. F., Ivy, G. O., and Brown, I. R. (1990) J. Neurosci. 34. Palmiter, R. D., Sandgren, E. P., Avarbock, M. R., Allen, D. D.,

Res. 25, 194–203. and Brinster, R. L. (1991) Proc. Natl. Acad. Sci. USA 88, 478–6. Laping, N. J., Teter, B., Nichols, N. R., Rozovsky, I., and Finch, 482.

C. E. (1994) Brain Pathol. 4, 259–275. 35. Neznanov, N. S., and Oshima, R. G. (1993) Mol. Cell. Biol. 13,7. Brenner, M. (1994) Brain Pathol. 4, 245–257. 1815–1823.8. Brenner, M., Lampel, K., Nakatani, Y., Mill, J., Banner, C., 36. Ash, J., Ke, Y., Korb, M., and Johnson, L. F. (1993) Mol. Cell.

Mearow, K., Dohadwala, M., Lipsky, R., and Freese, E. (1990) Biol. 13, 1565–1571.Mol. Brain Res. 7, 277–286. 37. Farnham, P. J., and Means, A. L. (1990) Mol. Cell. Biol. 10,

9. Balcarek, J. M., and Cowan, N. J. (1985) Nucleic Acids Res. 13, 1390–1398.5527–5543. 38. Sachs, A. B. (1993) Cell 74, 413–421.

10. Miura, M., Tamura, T., and Mikoshiba, M. (1990) J. Neurochem. 39. Kozak, M. (1994) Biochimie 76, 815–821.55, 1180–1188.

40. Kozak, M. (1989) J. Cell Biol. 108, 229–241.11. Sarid, J. (1991) J. Neurosci. Res. 28, 217–228.

41. Huang, M. T., and Gorman, C. M. (1990) Mol. Cell. Biol. 10,12. Kaneko, R., and Sueoka, N. (1993) Proc. Natl. Acad. Sci. USA 1805–1810.

90, 4698–4702.42. Mercer, E. H., Hoyle, G. W., Kapur, R. P., Brinster, R. L., and

13. Condorelli, D. F., Nicoletti, V. G., Barresi, V., Caruso, A., Conti- Palmiter, R. D. (1991) Neuron 7, 703–716.cello, S., de Vellis, J., and Stella, A. M. G. (1994) J. Neurosci.

43. Brenner, M., Kisseberth, W. C., Su, Y., Besnard, F., and Messing,Res. 39, 694–707.A. (1994) J. Neurosci. 14, 1030–1037.

14. Besnard, F., Brenner, M., Nakatani, Y., Chao, R., Purohit, H. J.,44. Tolentino, P. J., Dikkes, P., Tsuruda, L., Ebert, K., Fink, J. S.,and Freese, E. (1991) J. Biol. Chem. 266, 18877–18883.

Villa-Komaroff, L., and Lamperti, E. D. (1995) Mol. Brain Res.15. Kaneko, R., Hagiwara, N., Leader, K., and Sueoka, N. (1994) 33, 47–60.

Proc. Natl. Acad. Sci. USA 91, 4529–4533.45. Galou, M., Pourin, S., Ensergueix, D., Ridet, J.-L., Tchelingerian,

16. Miura, M., Tamura, T., and Mikoshiba, K. (1991) Dev. Growth J.-L., Lossouarn, L., Privat, A., Babinet, C., and Dupouey, P.Differ. 33, 111–116. (1994) Glia 12, 281–293.

17. Hatch, N., and Sarid, J. (1994) J. Neurochem. 63, 2003–2009. 46. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher,18. Sarkar, S., and Cowan, N. J. (1991) J. Neurochem. 57, 675–684. D. C. (1994) Science 263, 802–805.19. Masood, K., Besnard, F., Su, Y., and Brenner, M. (1993) J. Neuro- 47. Paldi, A., Deltour, L., and Jami, J. (1993) Transgenic Res. 2,

chem. 61, 160–166. 325–329.20. Feinstein, D. L., Weinmaster, G. A., and Milner, R. J. (1992) 48. Sokolov, B. P., Ala-Kokko, L., Dhulipala, R., Arita, M., Khillan,

J. Neurosci. Res. 32, 1–14. J. S., and Prockop, D. J. (1995) J. Biol. Chem. 270, 9622–9629.21. Galea, E., Dupouey, P., and Feinstein, D. L. (1995) J. Neurosci. 49. Furth, P. A., Onge, L. S., Boger, H., Gruss, P., Gossen, M.,

Res. 41, 452–461. Kistner, A., Bujard, H., and Hennighausen, L. (1994) Proc. Natl.Acad. Sci. USA 91, 9302–9306.22. Zelenika, D., Grima, B., Brenner, M., and Pessac, B. (1995) Mol.

Brain Res. 30, 251–258. 50. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W.,and Bujard, H. (1995) Science 268, 1766–1769.23. Nakatani, Y., Brenner, M., and Freese, E. (1990) Proc. Natl.

Acad. Sci. USA 87, 4289–4293. 51. Mucke, L., Oldstone, M. B. A., Morris, J. C., and Nerenberg,M. I. (1991) New Biol. 3, 465–474.24. Nakatani, Y., Horikoshi, M., Brenner, M., Yamamoto, T.,

Besnard, F., Roeder, R. G., and Freese, E. (1990) Nature 348, 52. Campbell, I. L., Abraham, C. R., Masliah, E., Kemper, P., Inglis,86–88. J. D., Oldstone, M. A. B., and Mucke, L. (1993) Proc. Natl. Acad.

Sci. USA 90, 10061–10065.25. Palmiter, R. D., and Brinster, R. L. (1986) Annu. Rev. Genet. 20,465–499. 53. Toggas, S. M., Masliah, E., Rockenstein, E. M., Rall, G. F.,

Abraham, C. R., and Mucke, L. (1994) Nature 367, 188–193.26. Bronson, S. K., and Smithies, O. (1994) J. Biol. Chem. 269,27155–27158. 54. Verderber, L., Johnson, W., Mucke, L., and Sarthy, V. (1995)

Invest. Ophthalmol. Vis. Sci. 36, 1137–1143.27. Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994)Manipulating the Mouse Embryo: A Laboratory Manual, 2nd 55. Reynolds, B. A., Tetzlaff, W., and Weiss, S. (1992) J. Neurosci.ed., Cold Spring Harbor Laboratory Press, Plainview, NY. 12, 4565–4574.

28. McKnight, R. A., Shamay, A., Sankaran, L., Wall, R. J., and 56. Lewis, S. A., and Cowan, N. J. (1985) J. Neurochem. 45, 913–Henninghausen, L. (1992) Proc. Natl. Acad. Sci. USA 89, 6943– 919.6947. 57. Tardy, M., Fages, C., Roi, H., LePrince, G., Rataboul, P., Char-

riere-Bertrand, C., and Nunez, J. (1989) J. Neurochem. 52, 162–29. Chung, J. H., Whiteley, M., and Felsenfeld, G. (1993) Cell 74,505–514. 167.



58. Nichols, N. R., Day, J. R., Laping, N. J., Johnson, S. A., and 75. Delaney, C. L., Brenner, M., and Messing, A. (1994) Soc. Neu-rosci. 20, 613.17 [Abstract].Finch, C. E. (1993) Neurobiol. Aging 14, 421–429.

76. Delaney, C. L., Brenner, M., and Messing, A. (1995) Soc. Neu-59. Bovolenta, P., Liem, R. K. H., and Mason, C. A. (1987) Dev. Brainrosci. 21, 319.19 [Abstract].Res. 33, 113–126.

77. Henderson, J. T., Seniuk, N. A., Richardson, P. M., and Roder,60. Hirano, M., and Goldman, J. E. (1988) J. Neurosci. Res. 21, 155–J. C. (1994) Soc. Neurosci. 20, 451.11 [Abstract].167.

78. Schwartz, J. P., Taniwaki, T., Messing, A., and Brenner, M.61. Teter, B., Rozovsky, I., Krohn, K., Anderson, C., Osterburg, H.,(1996) Ann. N.Y. Acad. Sci. 780, 29–35.and Finch, C. (1996) Glia, 17, 195–205.

79. Mucke, L., and Rockenstein, E. M. (1993) Transgene 1, 3–9.62. Schonrich, G., Kalinke, U., Momburg, F., Malissen, M., Schmitt-Verhulst, A.-M., Malissen, B., Hammerling, G. J., and Arnold, 80. Turnley, A. M., Morahan, G., Okano, H., Bernard, O., Mikoshiba,B. (1991) Cell 65, 293–304. K., Allison, J., Bartlett, P. F., and Miller, J. F. A. P. (1991) Nature

353, 566–569.63. Sarthy, V., and Egal, H. (1995) DNA Cell Biol. 14, 313–320.81. Allison, J., Campbell, I. L., Morahan, G., Mandel, T. E., Harrison,64. Holash, J. A., Sami, I., Harik, G. P., and Stewart, P. A. (1993)

L. C., and Miller, J. F. A. P. (1988) Nature 333, 529–533.Proc. Natl. Acad. Sci. USA 90, 11069–11073.82. Borrow, P., Cornell, J. L., Ruppe, M. D., and Mucke, L. (1995)65. Braun, R. E., Lo, D., Pinkert, C. A., Widera, G., Flavell, R. A.,

Soc. Neurosci. 21, 590.2 [Abstract].Palmiter, R. D., and Brinster, R. L. (1990) Biol. Reprod. 43, 684–83. Wyss-Coray, T., Borrow, P., and Mucke, L. (1995) Genetic Models493.

for Multiple Sclerosis and Related Disorders [Abstract].66. Al-Shawi, R., Burke, J. I., Wallace, H., Jones, C., Harrison, S.,84. Campbell, I. L. (1995) Int. J. Dev. Neurosci. 13, 275–284.Buxton, D., Maley, S., Chandley, A., and Bishop, J. O. (1991)

Mol. Cell. Biol. 11, 4207–4216. 85. Hickey, W. F., and Kimura, H. (1988) Science 15, 290–292.67. Wyss-Coray, T., Feng, L., Masliah, E., Ruppe, M. D., Lee, H. S., 86. Finch, C. E., Laping, N. J., Morgan, T. E., Nichols, N. R., and

Toggas, S. M., Rockenstein, E. M., and Mucke, L. (1995) Am. J. Pasinetti, G. M. (1993) J. Cell. Biochem. 53, 314–322.Pathol. 147, 53–67. 87. Kuruvilla, A. P., Shah, R., Hochwald, G. M., Liggitt, H. D., Pal-

68. Rall, G. F., Mucke, L., Nerenberg, M., and Oldstone, M. B. A. ladino, M. A., and Thorbecke, G. J. (1991) Proc. Natl. Acad. Sci.(1994) J. Neuroimmunol. 52, 61–68. USA 88, 2918–2921.

69. Steffensen, S. C., Campbell, I. L., and Henriksen, S. J. (1994) 88. Galbreath, E. J., Kim, S.-J., Brenner, M., and Messing, A. (1995)Brain Res. 652, 149–153. Am. College Vet. Pathol. [Abstract].

89. Constam, D. B., Schmid, P., Aguzzi, A., Schachner, M., and Fon-70. Chiang, C. S., Stalder, A., Samimi, A., and Campbell, I. L. (1994)tana, A. (1994) Eur. J. Neurosci. 6, 766–778.Dev. Neurosci. 16, 212–221.

90. McKinnon, R. D., Piras, G., Ida, J. A., Jr., and Dubois-Dalcq, M.71. Brett, F., Mizisin, A. P., Powell, H. C., and Campbell, I. L. (1995)(1993) J. Cell Biol. 121, 1397–1407.J. Neuropathol. Exp. Neurol. 54, 766–775.

91. Masu, Y., Wolf, E., Holtmann, B., Sendtner, M., Brem, G., and72. Chiang, C. S., Masliah, E., Stalder, A., Samimi, A., and Camp-Thoenen, H. (1993) Nature 365, 27–32.bell, I. L. (1994) Soc. Neurosci. 20, 176.14 [Abstract].

92. Winter, C. G., Saotome, Y., Levison, S. W., and Hirsh, D. (1995)73. Campbell, I. L., Chiang, C.-S., Powell, H. C., Roberts, A. J., andProc. Natl. Acad. Sci. USA 92, 5865–5869.Gold, L. H. (1995) Soc. Neurosci. 21, 590.6 [Abstract].

93. Schwartz, J. P. (1992) Int. Rev. Neurobiol. 34, 1–23.74. Campbell, I. L., Mucke, L., and Sandberg, K. (1993) Soc. Neu-rosci. 19, 98.2 [Abstract]. 94. Kolberg, R. (1994) J. NIH Res. 6, 62–64.


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