BAC transgene construction and characterization of Cre expression

To determine whether engineered BACs can be used generally to efficiently drive functional levels of constitutive or inducible Cre recombinases in specific CNS cell types, we have chosen to use 10 different BAC vectors to create transgenic lines expressing recombinase (Table 1). For the purpose of this study, BACs were selected from those previously characterized with BAC-EGFP constructs from the GENSAT project. Genes selected for this study represented those expressed selectively in neurotransmitter-specific populations, in specific components of forebrain neural circuits, or in specific neuron or glial populations. In each case, BAC engineering (Gong et al., 2002) was used to insert an intron containing Cre or CreERT2 cassettes, followed by a polyadenylation sequence to terminate transcription of the fusion transcript immediately after the recombinase gene, into the BAC vector at the initiating ATG codon in the first coding exon of the gene chosen for expression. This was done using a new shuttle vector (pLD53.SC-Cre) carrying the appropriate selectable markers and a small homology arm from the locus being targeted to guide recombination. An inducible recombinase driver line using CreERT2 (Indra et al., 1999) was also analyzed. Offspring of founders were crossed to a Rosa26/stop/EGFP reporter line (Soriano, 1999). Functional recombination of the reporter locus was analyzed for all lines using immunohistochemical detection of EGFP in adult mice carrying both the BAC construct and Rosa26/EGFP transgene. Because copy number, transgene insertion sites, and transient developmental expression of the chosen loci in previously undetected cell types could result in unexpected sites of recombinase activity, multiple transgenic founder lines for each gene were generated and analyzed. We report here that generation of Cre driver lines that express functional Cre in the expected cell types was achieved for all 10 engineered BAC vectors tested, including one inducible with tamoxifen. As predicted, in some cases there were variations in the patterns of Cre-induced EGFP expression, which are instructive in the general use of BACs to direct Cre expression in targeted neuronal and glial cell types. The patterns of functional Cre expression for 14 BAC-Cre transgenic lines are described.

Table 1.

List of BAC-Cre lines described in this study

Gene [BAC-Cre transgenic line(s)]	BAC clone	Cre expression pattern (adult/developmental/partial)
Choline acetyl transferase (Chat)	RP23-246B12
GM24/GM60		Adult
GM53		Partial: motor nuclei only
Tyrosine hydroxylase (Th)	RP23-350E13
FI12/FI172		Adult plus developmental pattern
Serotonin transporter (Slca4)	RP24-335M24
ET33/ET35		Adult
ET124		Adult plus developmental pattern
Noradrenalin transporter (Slc6a2)	RP23-109O23
FV317/FV319		Adult
ETS domain transcription factor (Etv1)	RP23-250K4
GM225		Neocortex layer 5
Neurotensin receptor 1 (Ntsr1)	RP23-314D14
GN220		Neocortex layer 6
D2 dopamine receptor (Drd2)	RP23-161H15
ER44		Adult
ER43		Partial: striatopallidal neurons
D1 dopamine receptor (Drd1a)	RP23-47M2
EY262/FK161/FK150		Adult
Purkinje cell protein (Pcp2)	RP24-186D18
GN135		Adult
Oligodendrocyte protein (Cmtm5)	RP24-317F19
GM160		Adult

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Neurotransmitter-specific genes

A mainstay of neuroscience research involves pharmacologic manipulation of specific neurotransmitter systems. Targeting genetic manipulations to neurons based on their neurotransmitter phenotype greatly expands approaches for manipulating such systems. For this purpose, four BAC-Cre constructs were used, which included two genes encoding neurotransmitter-synthesizing enzymes, tyrosine hydroxylase (Th) and choline acetyl transferase (Chat), and two genes encoding neurotransmitter transporters, the serotonin transporter (Slc6a4) and the noradrenalin transporter (Slc6a2). For two of these genes, Th and Slca4, Cre driver lines using knock-in approaches have been produced (Lindeberg et al., 2004; Zhuang et al., 2005), which allows for a comparison with the BAC-Cre constructs used here. For each gene, transgenic lines were produced with Cre specifically expressed in the appropriate adult neurotransmitter-specific neuron population as verified with double labeling for a protein marker specific for each neuron population (Fig. 1). In a Th BAC-Cre line (FI12), functional Cre expression is present in all of the catecholamine dopaminergic and noradrenergic neurons that express Th in the adult (for example, in the dopaminergic neurons in the substantia nigra pars compacta) (Fig. 1A). In a Slc6a4 BAC-Cre transgenic line (ET33), functional Cre is demonstrated in serotonin neurons in the dorsal and median raphe nuclei by colocalization of tryptophan hydroxylase immunoreactivity (IR) (Fig. 1B). Acetyl choline neurons display functional Cre in a Chat BAC-Cre line (GM24) as demonstrated by colocalization of Chat immunoreactivity in neurons in the basal forebrain (see Fig. 3A).

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Figure 1.

Cre expression directed to dopaminergic (A), serotonergic (B), and cholinergic (C) neurons in BAC-Cre transgenic mouse lines. A, Dopaminergic neurons are labeled in a Th BAC-Cre transgenic line with Cre expression localized in neurons in the midbrain substantia nigra pars compacta (Th BAC-CRE; green), which colocalize with Th-IR. There is a nearly complete overlap of TH Cre-driven EGFP labeling and Th-IR (insets). B, In a serotonin transporter (Slca4) BAC-Cre transgenic line, Cre is directed to serotonergic neurons in the dorsal raphe nucleus in the dorsal midbrain, which coexpress serotonin-synthesizing enzyme, tryptophan hydroxylase-IR. Insets, There is a nearly complete overlap of Slca4 Cre-driven EGFP labeling and tryptophan hydroxylase-IR in neurons in the dorsal raphe. C, In a Chat BAC-Cre transgenic line, labeled neurons in the basal forebrain coexpress Chat immunoreactivity (ChAT-IR).

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Figure 3.

A, A Pcp2 BAC-Cre transgenic line produces Cre expression that is restricted to the cerebellum localized to Purkinje cell neurons as seen in a sagittal brain section. B, A Cmtm5 BAC-Cre transgenic line produces Cre expression in oligodendrocyte cells throughout the brain. Bi, Labeled cells in a forebrain area including the cerebral cortex, underlying white matter, and striatum are seen to have the morphology of oligodendrocytes. C, Inducible Cre expression occurs in a serotonin transporter (Slca4) BAC-ER-Cre transgenic line. Mice treated with saline show no Cre expression, whereas a single treatment with tamoxifen (6 mg/kg, i.p.) produces Cre expression in serotonin neurons in the dorsal raphe.

A detailed mapping of Cre-induced EGFP in the brain for each of these transgenic lines is provided on the GENSAT website (http://www.gensat.org/index.html), and the distributions match the expression of the genes in the adult brain, for the most part. Variations in these lines and in other lines produced with the same BAC-Cre constructs for these genes are instructive. The two main types of variations encountered are those producing ectopic expression or partial expression. Ectopic expression, in which Cre-induced Rosa26 EGFP is observed in neurons that do not express the endogenous gene in the adult brain, was observed in the Th BAC-Cre lines and in some but not all of the Slc6a4 BAC-Cre lines. In two Th BAC-Cre lines (FI12 and FI172), Cre-induced EGFP expression is present in essentially all adult neurons in which Th-IR is present. Additionally, EGFP is also observed in selected neurons that are Th-IR negative in the adult, particularly in forebrain areas, including the striatum and hypothalamus (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). The two most likely causes of ectopic expression are insertion of the transgene construct into a locus that alters its expression and transient expression of the gene in neurons during development, which do not express the gene in the adult. In this case, several reasons point to the latter mechanism. First, multiple studies have demonstrated transient developmental expression of Th in the same forebrain areas seen in the Th BAC-Cre lines (Komori et al., 1991; Marin et al., 2005). Second, other transgenic Th-Cre lines produced with different transgene vectors using a knock-in approach produce patterns of ectopic Cre expression nearly identical to those seen in the Th BAC-Cre line, which were ascribed to transient developmental expression (Lindeberg et al., 2004; Zhuang et al., 2005). Moreover, the multiple Th BAC-Cre lines analyzed in this study produce identical ectopic expression patterns. Because Th BAC-Cre constructs likely insert into different loci in different lines, it is unlikely that different insertional loci would produce identical patterns of ectopic expression (Heintz, 2001). Similar variations of Cre expression were obtained with the Slca4 BAC-Cre construct. In two lines (ET33 and ET35), Cre expression was localized selectively to adult neurons using serotonin as a neurotransmitter, most notably serotonin neurons in the dorsal and median raphe (Fig. 1B). Another line (ET124) generated with this BAC-Cre construct displayed expression not only in adult serotonin neurons, but also in corticothalamic and thalamocortical neurons (supplemental Fig. 3, available at www.jneurosci.org as supplemental material). These latter neurons have been shown to express the serotonin transporter transiently during development (Lebrand et al., 1998) and have shown Cre expression using other Cre constructs (Zhuang et al., 2005). Thus, in this line, ectopic Cre-induced EGFP expression is likely caused by transient expression of the Slca4 gene during development. In the case of the ectopic Cre-induced EGFP expression seen in both the Th and Slca4 BAC-Cre lines, the pattern of expression is very specific and restricted to neurons expressing the endogenous genes transiently during development.

The second type of variation produces partial patterns of Cre-induced EGFP expression compared with the expression of the endogenous gene. Such a partial pattern of expression is produced in one of the Chat BAC-Cre transgenic lines. Two Chat BAC-Cre transgenic lines (GM23 and GM54) produced Cre-induced EGFP expression throughout the brain and spinal cord that matches the expression of Chat in the adult. Another Chat BAC-Cre line (GM60) produced Cre-induced EGFP expression, which is restricted only to motor neurons in the brainstem and spinal cord (supplemental Fig. 4, available at www.jneurosci.org as supplemental material). In this case, the more restricted expression is most likely caused by a reduced number of Chat BAC-Cre construct copies being incorporated into the genome compared with the line in which Cre is expressed in the full complement of cholinergic neurons in the brain. Evidence for this mechanism is somewhat empirical, based on the robustness of Cre-induced EGFP expression in the Chat BAC-Cre line labeling all cholinergic neurons. In these lines, EGFP labeling of forebrain neurons reveals only cell bodies and dendrites, whereas motor neurons are more robustly labeled, with axonal projections clearly labeled. This suggests that there are relative differences between forebrain cholinergic neurons and motor neurons in the level of expression of Chat. Thus, differences in Cre-induced EGFP expression between these lines may reflect the level of Cre expression, which is affected by the number of copies of the construct incorporated in the different lines.

Variations of Cre expression patterns produced with BAC-Cre drivers to target neurotransmitter-specific populations are instructive. On the one hand, Cre-driver lines have been produced with expression restricted to the target neuron populations in the adult. On the other hand, the variations in patterns produced with some lines demonstrate the need to carefully characterize the patterns of expression. In the case of ectopic expression caused by transient gene expression during development, there are several points to be made. First, such cases underscore the necessity of using a reporter line such as the Rosa26 EGFP line to characterize Cre driver lines. Such variations may produce greater specificity of Cre expression or may require use of other BAC-Cre driver lines to more specifically target particular neuron populations.

Genes expressed in specific forebrain circuits

Whereas targeting neuron populations based on neurotransmitter phenotype is useful for certain brain systems, other systems require a different strategy. Neural circuits in the forebrain provide such a case. For example, there are a large variety of subtypes of principal projection neurons in the cerebral cortex with distinct patterns of connections, which are organized into layers and cytoarchitecturally defined areas. Because all of these use glutamate as a neurotransmitter, targeting cortical neuron subtypes requires the selection of genes unrelated to their neurotransmitter. Inspection of the GENSAT BAC-EGFP lines identified a number of genes with expression restricted within the cerebral cortex to distinct layers. Two such genes, Etv1, encoding the transcription factor etv1 (an ETS-binding transcription factor, also known as ER81), and the neurotensin 1 receptor (Ntsr1), have been shown in BAC-EGFP transgenic lines to be localized respectively in layers 5 and 6 of the neocortex. There is a similar situation in the striatum, where all of the main subtypes of projection neurons use GABA as a neurotransmitter. In contrast with the cortex, striatal neuron projection subtypes are intermingled with each other. However, the two main subtypes, striatonigral and striatopallidal projection neurons, selectively express the Drd1a and Drd2 dopamine receptors, respectively (Gerfen et al., 1990). Thus, these genes provide good candidates to target Cre expression to specific forebrain circuits. Figure 2 shows the Cre-induced EGFP expression in BAC-Cre transgenic lines produced with these four genes. Directed Cre expression is produced in different BAC-Cre transgenic lines in layer 5 corticostriatal neurons (Etv1 BAC-Cre), in layer 6 corticothalamic neurons (Ntsr1 BAC-Cre), in striatopallidal neurons (Drd2 BAC-Cre), and in striatonigral neurons (Drd1 BAC-Cre). The robustness of Cre expression in these lines is such that the EGFP-immunohistochemical labeling produced with the EGFP reporter line clearly shows the specific axonal projections in each of these Cre driver lines.

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Figure 2.

Cre expression in forebrain circuits, with labeling of specific neuronal projection systems in the cerebral cortex (A, Etv1; B, Ntsr1) and striatum (C, Drd2; D, Drd1a). Expression is produced in layer 5 corticostriatal neurons in an Etv1 BAC-Cre line (A, Etv1). Cre is expressed in layer 6 corticothalamic neurons in an ntsr1 BAC-Cre line (B, Ntsr1). Clearly labeled are the projection axons of these neurons, which terminate in the dorsal thalamic nuclei. In the striatum, the majority of neurons are medium spiny projection neurons, which are evenly divided into striatopallidal (indirect pathway) and striatonigral (direct pathway) neurons, which selectively express the drd2 and drd1a dopamine receptors, respectively. Cre expression produced in Drd2 BAC-Cre lines (C, Drd2) is directed to striatopallidal neurons. In this line, labeled neurons in the striatum extend axons that terminate in the globus pallidus external segment (GPe). In contrast, expression produced in Drd1a BAC-Cre lines (D, Drd1a) is directed to striatonigral neurons, which have axons that extend through the globus pallidus to terminate in the internal segment of the globus pallidus (GPi) and substantia nigra (not shown).

The Etv1 and Ntsr1 BAC-Cre lines display expression that is restricted principally to the cerebral cortex, and within the cortex to neocortical areas and layers 5 and 6, respectively. In contrast, the Drd1a and Drd2 BAC-Cre lines, while producing specific expression in striatonigral and striatopallidal neurons, also produce expression in other brain regions. Such nonstriatal expression may provide some limitation for certain experiments compared with lines in which expression is restricted to striatal neurons. Another Drd2 BAC-Cre line (ER43) does have expression restricted primarily to striatopallidal neurons, with very little in other brain regions (supplemental Fig. 6, available at www.jneurosci.org as supplemental material). As with the partial expression in the Chat BAC-Cre line (GM60), the more restricted expression is most likely attributable to fewer copies of the BAC-Cre construct being inserted in the ER43 line than in the ER44 line. Again, the evidence for this is somewhat empirical in that it appears that there are differences in the level of Drd2 Cre-induced EGFP in different brain regions based on the robustness of labeling in axonal projections. A comparison of the Cre-induced EGFP labeling between these two lines shows an overall reduction in the ER43 line, which is likely caused by a dose effect. In this case, such reduced expression may provide for more selective targeting of striatopallidal neurons, compared with the line in which expression is more widespread.

Purkinje cell protein (Pcp2) and oligodendrocyte protein (Cmtm5) BAC-Cre transgenic lines

A major advantage of the use of BAC constructs to direct recombinase expression to specific neuron types is the general applicability to a large variety of gene loci. To further test this approach, two other genes expressed in either a specific neuron type, the Purkinje cell protein 2 (Pcp2) expressed specifically in the cerebellum by Purkinje cells and the CKLF-like MARVEL transmembrane dopamine-containing 5 protein (Cmtm5) expressed in oligodendrocytes were used to generate BAC-Cre driver lines. As shown in Figure 3, these lines produced specific Cre expression in the appropriate brain cell types.