Abstract
Genetic tools are enabling the molecular dissection of the functions and mechanisms of many biological processes. Transgenic manipulations provide powerful tools with which to test hypotheses regarding functions of specific cell types and molecules in vivo in combination with different types of experimental models. Various techniques are available that can target genetic manipulations specifically to astrocytes and that are enabling the molecular dissection of astrocyte biology in vivo. This article summarizes procedures and experience from our laboratory using transgenic strategies that enable either the ablation of proliferating astrocytes and related cells, or the deletion of specific molecules selectively from astrocytes, to study the functions of astrocytes and related cell types in health and disease in vivo using different experimental mouse models.
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1 Introduction
Astrocytes play essential roles in many different CNS functions including maintenance of the extracellular balance of ions, fluid balance and transmitters, provision of energy metabolites to neurons, participation in synaptic function and plasticity, and regulation of blood flow. Astrocytes also respond to all forms of CNS injury and disease with reactive astrogliosis and increasing evidence indicates that astrocytes have the potential to influence almost any CNS disorder either through loss or gain of functions (1, 2). There is ever growing interest in understanding the functions of astrocytes, the molecular mechanistic basis of those functions and how astrocytes influence neuronal function in both health and disease. Transgenic manipulations and other molecular techniques provide powerful tools with which to investigate astrocyte functions and mechanisms in healthy CNS and in experimental models of specific CNS insults. This article will describe two transgenic strategies that enable either the ablation of proliferating cells or the deletion of specific molecules selectively from specific cells by using selective targeting with cell-type specific promoters, and will discuss the experience from our laboratory using these strategies to study the functions of astrocytes and related cell types (see Note 1).
1.1 Strategy for Ablation of Proliferating Astrocytes and Related Cell Types
To transgenically target astrocytes and related cells we used a mouse glial fibrillary acid (mGFAP) promoter cassette consisting of the entire mGFAP gene plus flanking regions (see Subheading 2.1.1). This large promoter enabled faithful targeting of transgenes to cells expressing endogenous mGFAP as determined by extensive single cell analysis as described below. To selectively ablate proliferating astrocytes and related cell types (see Notes 1, 2). we targeted herpes simplex virus thymidine kinase (HSV-TK) to them using an mGFAP-TK transgene (see Subheading 2.1.1). Mammalian cells that express HSV-TK transgenes become selectively vulnerable to the antiviral agent, ganciclovir (GCV), while they are undergoing cell division (3–5). Expression of transgene-derived HSV-TK on its own has no detectable detrimental effects on mammalian cells in vivo or in vitro (5, 6). However, mammalian cells that express HSV-TK phosphorylate GCV, which is a thymidine analogue, and the phosphorylated GCV becomes trapped in TK expressing cells where it stops DNA replication during cell division, thereby killing cells via an apoptotic, noninflammatory mechanism (7). Our experiences using mGFAP-TK mice treated with different regimens of GCV delivery to selectively ablate proliferating astrocytes and other types of GFAP-expressing cells in a variety of experimental contexts are summarized below.
1.2 Strategy for Deletion of Specific Molecules Selectively from Astrocytes and Related Cell Types
To delete genes selectively from astrocytes and related cell types we used Cre-loxP technology (8) in combination with transgenic targeting via a large mGFAP promoter cassette (see Subheading 2.1.2). Cre recombinase (Cre) is an enzyme derived from P1 bacteriophage that catalyzes recombination between two 34-bp consensus sequences of DNA known as loxP sites. Cre-mediated recombination between loxP sites results in excision of the intervening DNA sequence. Cre acts efficiently in mammalian cells, and can be used to mediate recombination between loxP sites inserted into specific locations in order to delete targeted gene sequences. To do so, a target locus is flanked with loxP sites (floxed) in embryonic stem cells and mice are produced. Recombination can then be achieved in specific cell types in vivo by breeding these animals to mice that express Cre from selected promoters (8). Cre-mediated recombination between loxP sites can also be used to activate gene expression selectively in specific cells by deletion of STOP signals, and this approach can be used to conduct cell fate mapping and cell lineage studies (9). Our experiences using mGFAP-Cre mice crossed with either loxP-reporter mice for cell fate mapping studies or with loxP mice for selective gene deletion in astrocytes and other types of GFAP-expressing cells in a variety of experimental contexts are summarized below.
2 Materials
2.1 Gene Constructs and Plasmids
2.1.1 mGFAP-TK Plasmid
A mouse-GFAP-TK fusion gene construct was generated as described (5). Briefly, starting with a 15 kb mGFAP-lacZ plasmid, clone #445 (10), the lacZ sequence was replaced with HSV-TK sequence. This GFAP promoter cassette (clone #445) consists of a modified sequence of the entire murine GFAP gene that contains all introns, promoter regulatory elements, exons, and 2 kb of 3′ and 2.5 kb of 5′ flanking regions; expression of GFAP is prevented by the removal of a small fragment of the first exon (10). HSV-TK sequence, including a nuclear localization signal, artificial intron and polyadenylation signal (11), was modified by PCR to include Not I and Sal I 5′ and 3′ termini. The Not I – Sal I fragment was ligated into the first exon of the mGFAP promoter cassette. Integrity of the HSV-TK insert was confirmed by sequencing. The resulting mGFAP-TK plasmid is now available from Addgene plasmid 24703: GFAP-HSV-tk (pTGB008) http://www.addgene.org/Michael_Sofroniew.
2.1.2 mGFAP-Cre Plasmid
A mouse-GFAP-Cre fusion gene construct was generated as described (12). Briefly, starting with the mGFAP-HSV-TK plasmid described above in Subheading 2.1 (5), HSV-TK sequence was replaced with Cre recombinase sequence (12). The resulting mGFAP-TK plasmid is now available from Addgene, plasmid 24704: GFAP-Cre, http://www.addgene.org/Michael_Sofroniew.
2.2 Transgenic Mice
2.2.1 mGFAP-TK Transgenic Mice
mGFAP-TK transgenic mice line 7.1 were generated and characterized as described (4, 5). Briefly, linearized fusion gene construct derived from mGFAP-HSV-TK plasmid described in Subheading 2.1.1 was pressure injected into the male pronucleus of fertilized eggs from superovulated female mice and two-cell stage eggs are reimplanted into pseudopregnant foster mothers according to standard procedures. Founder lines were screened and line 7.1 was selected for use on the basis of its high penetrance and specificity of expression as demonstrated by cell characterization analyses that demonstrated greater than 98% of cells that express mouse GFAP also express TK expression and that 100% of TK expressing cells were mGFAP positive (4, 5, 13). As with many HSV-TK transgenic lines using different promoters, males are sterile (5, 14) and for this reason, experimental and control animals are obtained by mating heterozygous females with wild-type males (C57Bl6). Thus, transgenic and nontransgenic control animals are littermates with similar genetic backgrounds. The mGFAP-TK line 7.1 is fully (>10 generations) back-crossed onto a C57Bl6 background. The resulting transgenic mice are now available from Jackson Labs, JAX Stock# 005698 B6.Cg-Tg(Gfap-Tk)7.1Mvs/J; http://jaxmice.jax.org/strain/005698.html.
2.2.2 mGFAP-Cre Transgenic Mice Line 73.12
mGFAP-Cre transgenic mice line 73.12 was generated and characterized as described (12, 15) using the same mGFAP-Cre plasmid described in Subheading 2.1.2. Founder lines were screened and line 73.12 was selected for use on the basis of its high penetrance and specificity of expression as demonstrated by cell characterization analyses that demonstrated greater than 98% of cells that express mouse GFAP also express Cre or exhibit evidence of Cre activity and that 100% of Cre expressing cells were mGFAP positive (12, 15). Further characterization of the targeting specificity and selectivity of mGFAP-Cre line 73.12 to astrocytes and postnatal GFAP-expressing neural progenitor cells using Cre-reporter mice is described in Subheading 3.8. These mice breed well and are maintained by crossing heterozygous males or females with wild-type mates (C57Bl6). We prefer not to maintain homozygous animals because of potential developmental disturbances reported in certain transgenic lines expressing high levels of nuclear Cre recombinase in homozygous Cre mice (16). We have never noted any detectable abnormalities in heterozygous mGFAP-Cre mice. The mGFAP-Cre line 73.12 is fully (>10 generations) back-crossed onto a C57Bl6 background. The resulting transgenic mice are now available from Jackson Labs, JAX Stock# 012886 B6.Cg-Tg(Gfap-cre)73.12Mvs/J; http://jaxmice.jax.org/strain/012886.html.
2.2.3 mGFAP-Cre Transgenic Mice Line 77.6
mGFAP-Cre transgenic mice line 77.6 was generated similarly to line 73.12 using the same mGFAP-Cre plasmid described in Subheading 2.1.2. Founder lines were screened and line 77.6 was selected for use on the basis of its high penetrance and specificity of expression as demonstrated by cell characterization analyses that demonstrated greater than 98% of cells that express mouse GFAP also express Cre or exhibit evidence of Cre activity and that 100% of Cre expressing cells were mGFAP positive. Further characterization of the targeting specificity and selectivity of mGFAP-Cre line 77.6 to astrocytes but few postnatal GFAP-expressing neural progenitor cells using Cre-reporter mice is described in Subheading 3.8. These mice breed well and are maintained by crossing heterozygous males or females with wild-type mates (C57Bl6). We prefer not to maintain homozygous animals for reasons discussed in Subheading 2.2.2. The mGFAP-Cre line 77.6 is fully (>10 generations) back-crossed onto a C57Bl6 background. The resulting transgenic mice are now available from Jackson Labs, JAX Stock# 012887 B6.Cg-Tg(Gfap-cre)77.6Mvs/J; http://jaxmice.jax.org/strain/012887.html.
2.2.4 ROSA-β-Galactosidase Reporter Mice
Mice that express the reporter molecule β-galactosidase from the bacterial lacZ gene under the control of the ROSA (R26R) promoter and incorporating a loxP flanked “STOP” sequence (9) were purchased from Jackson Labs (JAX). JAX catalogue number: http://jaxmice.jax.org/strain/010633.html.
2.3 PCR Primers for Genotyping Transgenic Mice
2.3.1 PCR Primers for mGFAP-TK Transgenic Mice
The primer sequences used to genotype the mGFAP-TK mice are:
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5′-CGA GGC GGT GTT GTG TGG TGT-3′
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5′-GGT CCC GGA TCC GGT GGT GG-3′
2.3.2 PCR Primers for mGFAP-Cre Transgenic Mice
The primer sequences used to genotype the mGFAP-Cre mice are:
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5′-CCG GTT ATT CAA CTT GCA CC-3′
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5′-CTG CAT TAC CGG TCG ATG CAA C-3′
2.3.3 PCR Primers for ROSA-β-Galactosidase Reporter Mice
The primer sequences used to genotype the ROSA-β-galactosidase reporter mice are:
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5′-CGG TGA TGG TGC TGC GTT GG-3′
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5′-GAA TCA GCA ACG GCT TGC CG-3′
2.4 Ganciclovir
Our studies routinely use clinical grade GCV in the form of Cytovene i.v. (Hoffman La Roche, USA) or Cymevene (Roche, Europe). Other preparations of GCV that are not sodium salts and are not water-soluble are not useful for in vivo studies.
3 Methods
3.1 Mouse Care, Housing, and Genotyping
Mice are housed in a 12 h light/dark cycle in an SPF facility with controlled temperature and humidity and allowed free access to food and water, and all experiments and surgical procedures were conducted according to protocols approved by the Chancellor’s Animal Research Committee of the Office for Protection of Research Subjects at UCLA. Mice are genotyped by PCR analysis of DNA extracted from tissue samples (tail or ear snips) using the primers described above (see Subheading 2.3) and standard procedures: http://jaxmice.jax.org/support/genotyping/resources.html.
3.2 Ganciclovir (GCV) Delivery Regimens
We found that peripherally administered GCV was successful in ablating proliferating mGFAP-TK expressing cells in both the healthy and injured CNS (4, 12, 13, 17, 18). GCV crosses the blood brain barrier such that cerebrospinal fluid (CSF) levels are about 30% of serum levels (Roche data for Cymevene or Cytovene). We have identified several different peripheral delivery protocols of GCV that are successfully able to ablate proliferating cells that express the mGFAP-TK transgene and that are useful for different types of experimental contexts.
3.2.1 Continuous Subacute (7 Day) Delivery of High Dose GCV via Subcutaneous (SC) Osmotic Minipump
To deliver GCV continuously, we used osmotic minipumps (Alzet, Cupertino, CA) implanted subcutaneously according to the manufacturer’s instructions (4, 5). We found that a high dose of GCV administered continuously at a rate of 100 mg/kg/day in sterile physiological saline via such subcutaneous osmotic minipumps for the first 7 days after surgery was sufficient to ablate the majority of proliferating reactive astrocytes that form scars around traumatic injuries in the brain or spinal cord (4, 13). Practically this dose was achieved by dissolving 500 mg of Cytovene in 1.6 mL of sterile saline and administered in 7 day osmotic minipump at a rate of 0.5 μL h for an average mouse of 30 g. Given the excess nature of the GCV dose, variability of mouse weight was not found to influence efficacy. It should be noted that continuous delivery of GCV at this high dose (100 mg/kg/day) for greater than 12 days invariably led to a rapidly lethal inflammatory bowel syndrome due to ablation of GFAP-expressing enteric glia in the distal small intestine (5, 19). Bowel inflammation was never observed with GCV delivery of 7 or fewer days.
3.2.2 Continuous Chronic (3–6 Week) Delivery of Low Dose GCV via Sub Cutaneous (SC) Osmotic Minipump
For certain experimental contexts we became interested in delivering GCV for longer periods than 7 days, so we tested various deliver regimens and various doses of GCV delivery to mGFAP-TK mice. We found that a low dose of GCV administered continuously at a rate of 10 mg/kg/day in sterile physiological saline via such subcutaneous osmotic minipumps for 21 (12) or up to 42 days (20, 21) to otherwise healthy, uninjured mGFAP-TK mice was sufficient to ablate the majority of proliferating GFAP-expressing adult neural stem cells (NSCs) in the subependymal or subventricular zone of the lateral ventricles and in the subgranular zone of the hippocampal dentate gyrus (12, 20, 21). This low dose of continuous GCV delivery did not cause the inflammatory bowel gut illness observed with the high dose GCV delivery described above.
3.2.3 Intermittent Chronic (28 Day) Delivery of Low Dose GCV via Single Subcutaneous (SC) Injections Daily or Every Other Day
For other experimental contexts we became interested in delivering GCV for prolonged periods via single daily or every other day subcutaneous injections. We found that a low dose of GCV administered in sterile physiological saline as a single subcutaneous injection given every second day at 25 mg/kg/every other day for 28 days was effective in ablating the proliferating reactive astrocytes that form scar-like accumulations around perivascular cuffs of infiltrating leukocytes during adaptive immune inflammation in the CNS triggered by autoimmune encephalomyelitis (EAE) (18). In addition, we have found single daily injections of GCV at 25 mg/kg/every day for 7 days effective in ablating proliferating scar-forming astrocytes after CNS traumatic injury. These low doses of intermittent single injections of GCV did not cause the inflammatory bowel illness observed with the high dose GCV delivery described above.
3.3 mGFAP-TK Mediated Ablation of Proliferating Reactive Astrocytes After CNS Insults
Expression of transgene-derived HSV-TK on its own has no detectable detrimental effects on astrocytes in vivo or in vitro, and mGFAP-TK mice develop and function normally (4, 5). Astrocyte cell division is rare in the uninjured CNS (4, 5). Thus, in uninjured GFAP-TK mice GCV does not kill astrocytes, the majority of which are not mitotic, but after CNS injury in GFAP-TK mice GCV kills dividing reactive astrocytes in the immediate vicinity of the injury while sparing the nondividing astrocytes throughout the rest of the CNS. Using this model, we have studied the roles of reactive astrocytes scar-forming astrocytes after a variety of different types of CNS insults (4, 12, 13, 17, 18).
Reactive astrocytes that proliferate are an essential component of the glial scars that form in response to various types of CNS insults. In response to CNS trauma or stroke, reactive scar-forming astrocytes proliferate extensively in the immediate vicinity of the lesion, with a peak division time from 3 to 5 days after the insult (22, 23). We have found that the majority of these dividing GFAP-expressing, scar-forming astrocytes are ablated in mGFAP-TK mice by GCV administered for the first 7 days after the insult (see Subheading 3.1) (4, 13). In response to autoimmune inflammatory challenge, reactive astrocytes proliferate extensively in the immediate vicinity of certain blood vessels and form perivascular scar-like structures (18). We have found that the majority of these dividing GFAP-expressing, scar-forming astrocytes are ablated in mGFAP-TK mice by GCV administered chronically during EAE (see Subheading 3.1) (18).
3.4 mGFAP-TK Mediated Ablation of Proliferating GFAP-Expressing Neural Stem Cells in Uninjured Adult CNS or In Vitro
The postnatal and adult CNS contains populations of neural stem or progenitor cells (NSC) that express GFAP and are related to developmental radial progenitor cells (24, 25). We have found that the majority of these dividing GFAP-expressing, adult NSC can be ablated in vivo in mGFAP-TK mice by GCV chronic low dose GCV administered for at least 21 days in adult mice (see Subheading 3.1) allowing experimental studies on the functions of these cells (12, 20, 21). These postnatal NSC can also be selectively ablated in vitro in neurosphere cultures using GCV (26, 27).
3.5 Use and Comparison of mGFAP-Cre Lines 77.6 and 73.12 to Target Cre Activity to Astrocytes Alone or Astrocytes Plus Postnatal GFAP-Expressing Progenitors
We generated and extensively evaluated multiple founder lines of mGFAP-Cre mice generated using the same gene construct (see Subheading 2.1.2), each with their own particular pattern of Cre expression and activity as revealed by crossbreeding with Cre-reporter mice. Based on this screening, we identified and saved two founder lines with slightly differing expression patterns that we thought would be useful for different targeting purposes. These lines were designated line 73.12 (see Subheading 2.2.2) and line 77.6 (see Subheading 2.2.3) and both have been extensively characterized for Cre expression and targeting using reporter mice and double labeling studies evaluated at the single cell level. Both of these lines exhibit Cre expression patterns that faithfully mimic endogenous mouse GFAP-expression in over 98% of GFAP-expressing astrocytes in healthy CNS and in reactive astrocytes after different CNS insults. In this regard, it should be noted that not all astrocytes express GFAP in the healthy CNS, but all do so after CNS injury. The difference between the two lines is in their targeting of postnatal and adult neural stem or progenitor cells. Line 73.12 faithfully mimics endogenous mouse GFAP-expression in over 98% of postnatal and adult radial NSC in the subependymal or subventricular zone of the lateral ventricles and in the subgranular zone of the hippocampal dentate gyrus that give rise to new neurons throughout life (12, 15). In addition, line 73.12 faithfully mimics endogenous mouse GFAP-expression in various small subpopulations of late developing radial NSC that adopt GFAP expression while still giving rise to the last born neurons during postnatal development. In this later context, Cre activity and reporter gene expression are targeted to small subsets (less than 3% of cells) of the last developmentally born neurons that are scattered in layers 2 and 3 of the cerebral cortex (see Fig. 1a), hippocampal pyramidal layer (see Fig. 1c), cerebellar granular layer (see Fig. 1e), and various locations in the hypothalamus and midbrain, but not in the spinal cord (12, 15). In contrast, line 77.6 exhibits Cre targeting only to a subpopulation of neural progenitors in the adult subventricular zone (28) and appears to have no detectable targeting to neuronal progenitors in cerebral cortex, hippocampus, or cerebellum (see Fig. 1b, d, f ) (28). Thus, by combining high levels of targeting to astrocytes, with no targeting to neural progenitors in most CNS regions, line 77.6 is likely to be particularly useful for conditional molecular knockout (CKO) targeted specifically to astrocytes. We have found that both founder lines have faithfully transmitted their particular expression patterns through many generations.
3.6 mGFAP-Cre-loxP Mediated Deletion of Molecules from Astrocytes, Reactive Astrocytes, and Other GFAP-Expressing Cell Types
Using the mGFAP-Cre lines 73.12 and 77.6, our laboratory and other laboratories have successfully deleted various different types of molecule from GFAP-expressing cells to achieve conditional gene knockout (CKO) in astrocytes (15, 29, 30), NSC (12), and Schwann cell progenitors (31). The combination of this type of CKO with specific in vivo experimental models provides powerful tools with which to investigate the roles of these cells and of specific molecules that they express, in a variety of biological processes related to health and disease (see also Notes 3, 4 and 5).
3.7 mGFAP-Cre-loxP Mediated Lineage Analysis of Progeny of Adult Neural Stem Cells
Cre-loxP technology is useful not only for deleting molecules selectively from specific cell types, but can also be used for fate mapping and lineage analysis of cells derived from stem or progenitor cells. We have used mGFAP-Cre line 73.12 to demonstrate that GFAP-expressing NSC are the predominant source of constitutive neurogenesis in adult forebrain (12). These mice are similarly useful for investigating questions regarding the lineage of newly generated cells after CNS insults, or for fate mapping the potential of GFAP-expressing NSC after CNS insults (32).
3.8 Targeting of TK or Cre to GFAP-Expressing Cells in Peripheral Nerve and Enteric Nervous System
In addition to being expressed in CNS astrocytes and related cells, GFAP is expressed in various other tissues, particularly in glia of the peripheral and enteric nervous systems (33, 34). In the peripheral nervous system, Schwann cells transiently express GFAP during development and reacquire GFAP expression during the response to peripheral nerve injury. We have found that 7 days of high dose GCV (see Subheading 3.2.1) given to mGFAP-TK mice after peripheral nerve crush or transection is sufficient to ablate proliferating Schwann cells for studies of the effects of these cells on axon regeneration (35). We have found that 10 days of high dose GCV (see Subheading 3.2.1) given to mGFAP-TK mice is sufficient to ablate GFAP-expressing enteric glia, leading to severe tissue inflammation (5). We have also found that mGFAP-Cre mice target Cre activity to developing Schwann cells in peripheral nerves and can effectively mediate gene deletion in these cells for experimental studies (31).
4 Notes
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1.
Specificity of transgenic models and of the mGFAP lines described here
We have extensively characterized our transgenic mouse model for mGFAP-TK and mGFAP-Cre for targeting specificity and efficacy at the single cell level using combined, multicolored histological and immunohistochemical detection procedures evaluated using confocal microscopy (5, 12, 13, 15). This type of evaluation at the single cell level is essential for every transgenic founder line used for experimental purposes and should be conducted independently for every anatomical region studied using those lines. This type of detailed analysis is necessary because every transgenic line exhibits its own specific pattern of targeting due to stochastic insertion of the transgene into genome sites that may influence expression patterns. It is essential to understand the targeting pattern of any transgenic founder line in order to use that line effectively and appropriately. It is also well documented that transgenic targeting tends to be stably transmitted within individual transgenic founder lines (36), and we have also found this to be the case. Thus, once characterized, transgenic lines become useful tools for specific purposes.
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2.
mGFAP can target various cell types including astrocytes and related CNS cells, adult NSC, peripheral glial in gut and nerve
It is important to note that while GFAP is a molecule associated with most, if not all, reactive astrocytes in the CNS, it is not expressed at detectable levels by many astrocytes in the healthy CNS. It is also important to note that GFAP is not an exclusive marker for astrocytes, and is expressed by a variety of related cell types in the CNS, as well as glial cells in the peripheral and enteric nervous system, and by certain cells in the liver, kidney, pancreas, and other organs (5). As discussed above, the targeting of some of these cells in transgenic mice generated with mGFAP promoter can be useful for experimental purposes. Conversely, the targeting of other GFAP-expressing cell types needs also to be considered as a potential confound when evaluating the results of experiments targeted at one specific group. The targeting of various cell types does not negate usefulness of mGFAP transgenic models, which are considerably more selective than global gene deletion, and which compare favorably with other means of targeting reactive astrocytes and related cells when used correctly.
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3.
Comparison of different strategies for targeting Cre activity to astrocytes
Several different promoters are now in use for targeting transgenes to astrocytes, including mGFAP, hGFAP, GLAST, and Aldh1L1, and some of these have been combined with temporal regulation using doxycycline or tamoxifen-based inducible promoter systems. Each of these approaches has advantages and disadvantages that may make them more or less useful for specific experimental contexts. Understanding the targeting achieved by a specific promoter is essential to applying it appropriately. A small fragment of the human GFAP promoter (hGFAP) in common use is expressed robustly not only by mature astrocytes but also by early developmental radial progenitors and consequently targets Cre-mediated gene deletion to a substantial proportion of forebrain neurons (37). While useful to study the lineage of neurons derived from these early radial progenitors (38), this technique is not suitable for targeting conditional gene deletion selectively to astrocytes. Endogenous mouse GFAP (mGFAP) does not appear to be expressed by radial cell progenitors until the peri- and postnatal periods, and large promoter constructs of the mouse GFAP gene (mGFAP) can target Cre-mediated gene deletion exclusively to astrocytes in the spinal cord and other brain regions alone or in combination with adult neural progenitors depending on the individual transgenic line as discussed above. The molecule Aldh1L1 has recently been identified as a potentially useful astrocyte marker and means of transgenically targeting astrocytes with reporter molecules or Cre (39).
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4.
Inducible transgene activation by doxycycline or tamoxifen.
Temporal regulation of gene expression can be achieved using doxycycline or tamoxifen systems and these have been applied to astrocyte targeting using various promoters including hGFAP and mGFAP (40, 41) and the astrocyte glutamate transporter GLAST (42). Penetrance of inducible transgene activation by doxycycline or tamoxifen systems is generally only partial and can vary considerably from individual to individual. For some experimental purposes, partial penetrance can be an advantage by allowing the study of subsets of intensely labeled individual cells.
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5.
Considerations when using transgenic strains.
It is important to remember that there are caveats for all transgenic models. Targeting specificity and efficiency must be confirmed at the single cell level for every region studied using multiple labeling techniques as discussed above. With regard to doxycycline or tamoxifen-induced expression, it is important to realize that the variability of induction efficacy among individual mice can complicate quantitative comparisons across different experimental groups. In addition, as regards injury studies, tamoxifen has potent antiestrogenic effects that may impact on the response to traumatic injury, stroke, or inflammation (43). Each of the systems has pros and cons and it is important to understand their appropriate uses and limitations.
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Acknowledgments
The author’s work is supported by grants from the National Institutes of Health (NINDS) NS057624, Wings for Life, Multiple Sclerosis Society, and Adelson Medical Research Foundation.
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Sofroniew, M.V. (2012). Transgenic Techniques for Cell Ablation or Molecular Deletion to Investigate Functions of Astrocytes and Other GFAP-Expressing Cell Types. In: Milner, R. (eds) Astrocytes. Methods in Molecular Biology, vol 814. Humana Press. https://doi.org/10.1007/978-1-61779-452-0_35
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