WO1999043783A2 - TRANSGENIC MOUSE HAVING A LacZ REPORTER GENE UNDER THE CONTROL OF THE N-CAM PROMOTER - Google Patents

Abstract

The invention describes a transgenic mouse having a knockout N-CAM allele with an insertion of a reporter gene under the control of the N-CAM promoter. In preferred embodiments, the reporter gene encodes beta-galactosidase. The invention also describes methods for detecting modulators of the N-CAM gene using the transgenic mouse, or cultured cells obtained from the mouse.

Description

TRANSGENIC MOUSE HAVING A LacZ REPORTER GENE UNDER THE CONTROL OF THE N-CAM PROMOTER USEFUL FOR IDENTIFYING N-CAM MODULATORS, METHODS AND COMPOSITIONS

Technical Field

The invention relates a transgenic mouse having a N-CAM reporter gene construct useful for screening for N-CAM modulators, and to methods and compositions related to the transgenic mouse .

Background

Cell adhesion molecules (CAMs) mediate neuronal and glial adhesion during development of the nervous system and thereby affect processes such as neurite fasciculation, axonal pathfinding, and synaptogenesis . Edelman et al, Annu . Rev . Biochem.. 60:155-190 (1991); and Edelman, G. M. , Dev. Dynam.. 193:2-10 (1992). It has been suggested that in the adult brain, regulation of adhesion alters the morphological and physiological properties of synapses through effects on membrane interactions. Lynch et al, (1991) In (Ascher, P., Choi, D. . & Christen, Y. , eds . ) . Springer-Verlag Berlin Heidelberg, pp. 45-60; Fields et al, TINS. 19:473-480 (1996); and Kiss et al, Curr. Opin. Neurobio .. 7:640-646 (1977) . Changes in the levels of expression of CAMs may contribute to the maintenance of synaptic contacts and may be relevant to adult forms of activity-dependent synaptic plasticity. The question therefore arises as to what extent CAMs are regulated at the level of either transcription or translation by synaptic activity. The neural cell adhesion molecule, N-CAM, in particular participates in neuronal and astrocyte development in the central nervous system. The expression of N-CAM is therefor important from both the perspective of regulation of the gene encoding N-CAM and for the nature of the signaling pathways which affect N-CAM expression. In addition, because changes in various neuronal tissue expression of N-CAMs affects neurite fasciculation, axonal pathfinding and synaptogenesis, it is useful to have methods for the study of tissue distribution of expression of N-CAM.

Brief Summary of the Invention

The invention provides a system for characterizing N-CAM expression in vivo, for identifying tissue distribution of N-CAM expression under various physiologic, psychomotor and/or chemical stimuli, and for screening for bioactive molecules which modulate N-CAM expression.

The invention describes a transgenic mouse having a reporter gene under the control of the N-CAM promoter such that regulation of the N-CAM gene is reflected by regulated expression of the reporter gene in vivo . which reporter gene is convenient to assay. The N-CAM gene in the transgenic mouse has been modified by the insertion of the reporter gene between the transcription and translation initiation sites of the N-CAM gene of the mouse. The insertion places the expression of the reporter gene under the control of the N-CAM promoter. In preferred embodiments, the reporter gene is the bacterial lacZ gene. Using standard technology for the preparation of a transgenic mouse, the inserting is made in a single allele of the N-CAM gene, and through genetic crossing, one can produce a progeny mouse with the insertion in either a single N-CAM allele, i.e., heterozygous, or in both N-CAM alleles, i.e., homozygous .

The invention also describes methods for use of the transgenic mouse. As shown in the Examples, the transgenic mouse can be used to study tissue distribution of N-CAM expression by various analytical methods including in situ histological analysis of the expression pattern of the reporter gene, and in vitro detection of the reporter gene in various tissues, tissues sections and/or cell types.

The invention further contemplates a cultured cell derived from a transgenic mouse of the invention. The cultured cell can be obtained directly from the mouse, from a descendant mouse, or can be a progeny of a primary culture of one or more cells of a mouse of this invention. The cultured cell can be in the form of a single cell or cell line, or a composition of mixed cells. The cultured cell can be obtained from a mouse which is the descendant of a transgenic mouse of this invention, such as by a cross with another mouse having either the same or a different genetic background. Thus, a cell in the culture can be either homozygous or heterozygous for a reporter gene in the N-CAM allele.

A cultured cell of this invention has a variety of uses. For example, the cell can be used in vitro to study substances which affect N-CAM expression at the level of transcription or translation of the gene product. Other uses will be apparent to one skilled in the art. Methods for culturing a cell from a mouse of this invention, and for using such a cultured cell, are also described.

Brief Description of the Drawings Figures 1A-1E illustrate the overall strategy and details of targeted replacement of the N-CAM allele with lacZ, together with analysis of N-CAM expression in wild type, heterozygous and homozygous N-CAM knockout mice.

Figure 1A is a schematic diagram of the N-CAM locus in heterozygous animals after homologous recombination. In heterozygous mice, N-CAM promoter activity can be assayed both by β-gal activity and by quantitating N-CAM mRNA.

Figure IB illustrates the structures of the N-CAM targeting vector, wild-type N-CAM allele, and disrupted N-CAM allele. Restriction enzyme sites are abbreviated as: E, EcoRI ; K, Kpnl; N, NotI; S, Sail; Sa, SacII; X, Xhol . The hatched box above the diagram of the disrupted N-CAM allele indicates probe a (a Kpnl-Xhol intron 1 fragment) used for Southern blot analysis of the ES cell lines.

Figure IC illustrates a Northern blot. Total RNA isolated from the brains of wild type (+/+) , heterozygous N-CAM knockout (+/-) , and N-CAM knockout (-/-) mice was hybridized to a 300 bp probe for N-CAM.

Figure ID illustrates an ethidium bromide stain of the Northern gel of Figure IC prior to transfer and shows approximately equal loading of the total RNA.

Figure IE illustrates a Western blot of 30 μg total protein isolated from the brains of wild type (+/+), heterozygous (+/-) and homozygous (-/-) N-CAM knockout mice with a polyclonal antibody to N-CAM. Figures 2A-2H illustrate β-gal expression in heterozygous and homozygous knockout mice in whole mount embryos and in tissue sections compared with N-CAM mRNA localization. Whole mounts of heterozygous (+/-; Figures 2A & 2C) and of homozygous (-/-; Figures 2B & 2D) N-CAM knockout embryos, stained for β-gal expression at E9.5 (Figures 2A & 2B) and E13.5 (Figures 2C &

2D) , expressed β-gal throughout the spinal cord and dorsal root ganglia. Homozygous E9.5 embryos appeared to express a greater amount of β-gal than heterozygotes (Figure 3B) and β-gal activity quantitated by enzymatic assay was two fold higher in homozygotes, possibly reflecting a gene dosage effect. At E13.5 there was increased β-gal expression in the brain. Figures 2E & 2G illustrate sections of β-gal stained E13.5 embryos. β-gal expression was localized throughout the spinal cord and in the dorsal root ganglia (Figure 2E) . In the brain, β-gal was expressed in the post-mitotic neurons of the hindbrain and the midbrain as well as the in the floor plate (Figure 2G) . Figures 2F & 2H illustrate in si tu hybridization of E13.5 sections with N-CAM RNA probes. The expression of β-gal was similar to the pattern of N-CAM mRNA expression visualized by in si tu hybridization. sc, spinal cord; drg, dorsal root ganglia; fp, floor plate; mb, midbrain; hb, hindbrain.

Figures 3A-3G illustrate hippocampal morphology and physiology. Figures 3A & 3B illustrate morphology of adult hippocampus using a hematoxylin and eosin stain of wild type (Figure 3A, +/+) and homozygous (Figure 3B, -/-) adult brain. Figure 3C illustrates delivery of theta-burst stimulation (TBS) to path A (orthodromic) resulted in a rapid enhancement of fEPSP amplitudes that decayed over a brief period to a stable plateau. Approximately 1 hr later, application of the same high frequency stimulus to pathway B (antidromic) resulted in a comparable enhancement, without affecting established LTP in path A. The initial period of enhancement after TBS is thought to reflect a mixture of post-tetanic potentiation and short-term potentiation, a more decremental form of NMDA receptor-dependent plasticity. The brief heterosynaptic depression observed after TBS is likely related to adenosine release accompanying high-frequency stimulation, an effect commonly seen in hippocampal slices from normal mice.

Figure 3D illustrates an example of fEPSPs elicited with paired stimulation; interpulse interval equals 25msec. Figure 3E shows waveforms that are sample fEPSPs collected immediately before (top) and 40 min after (bottom) TBS. (scale bar : x = 10 ms , y = l mV) .

Figure 3F illustrates a typical record of LTP induced in slices from knockout mice. Plotted in the upper and lower panels are amplitudes of fEPSPs elicited alternately at 0.1Hz along two independent pathways in the stratum radiatum of field CA1.

Figure 3G illustrates a plot of the average amount of LTP obtained in groups of slices from N-CAM knockout and wild type mice. TBS-induced potentiation, expressed as a percentage of the pre-conditioning EPSP slope, was similar in the two groups of slices with regard to time course, magnitude, and input specificity (n = number of animals) .

Figures 4A-4B illustrates ampakine induction of β-gal expression resulting from N-CAM promoter activity in heterozygous N-CAM knockout mice. β-gal expression was examined in sagittal sections from vehicle-treated control (Figure 4A) and ampakine-treated (Figure 4B) mice. Ampakine injection increased β-gal expression in the CA1 and CA2 of the hippocampus and in the deep layers of the cortex.

Detailed Description of the Invention

A. Transgenic Mouse Having a Knock-Out N-CAM Allele

The invention describes a transgenic mouse which contains an exogenous gene (i.e, a "transgene") in its genome which is propagated in the genome when the animal is reproduced by any means, including by cloning or by the more common sexual crossing to produce progeny. A knockout mouse is a transgenic mouse wherein a preselected allele of the genome has been inactivated by some mechanism, typically by substituting a functional gene with a non-functional gene at that allele by homologous recombination or other mutagenic means.

In the present invention, a transgenic mouse is described having a reporter gene inserted into a region of the N-CAM gene of a chromosomal N-CAM allele of the mouse, wherein the region of insertion is located between a transcription start site and a translation start site in the N-CAM gene, and thereby prevents normal expression of the N-CAM structural gene under the control of the N-CAM promoter. Instead, the inserted reporter gene is expressed under the control of the N-CAM promoter in the knockout allele.

The inserted reporter gene is inserted by homologous recombination into the wild-type chromosomal N-CAM allele, and therefore can be engineered to recombine at any of a variety of positions in the N-CAM allele so long as the inserted reporter gene is positioned such that expression of the reporter gene is under the control of the N-CAM promoter. The positioning of the reporter gene into the chromosomal N-CAM allele is a particularly preferred aspect of the invention because it assures that the reporter/knockout construct in the mouse genome is in the native "landscape" of genetic elements of the N-CAM allele. The native landscape is important because it is not well understood how many different genetic elements in the region of the N-CAM allele participate in regulating N-CAM gene expression, nor is it understood how distant from the N-CAM structural gene those elements may be which can participate in the regulation. Thus, expression of the reporter gene in the transgenic mouse most accurately reflects as a model the N-CAM promoter activity and regulation of the N-CAM gene when using the invention for screening to identify modulators of N-CAM gene expression because the reporter gene is inserted into the native landscape of the chromosomal N-CAM gene. The inserted reporter gene can be placed at a variety of positions in the N-CAM allele and achieve the desired result, so long as the reporter gene is expressed under the control of the N-CAM promoter. For example, the insertion can be located at the N-CAM allele transcription initiation site, can be located at the N-CAM allele translation initiation site, or at any position in between. An exemplary construct is described in the Examples .

The reporter gene expresses a structural gene product which can be detected in the mouse by some means, and a variety of reporter genes are suitable for use in the invention. Exemplary reporter genes include genes which encode the green fluorescent protein (GFP) , the luciferase enzyme, chloramphenicol acetyltransferase, beta galactosidase, and the like proteins. These genes are readily available and the methods for their detection are well known, and therefore, the invention is not to be construed as so limited to any particular reporter gene. For example, the luciferase encoding gene is available in the plasmid pGL3 -Basic from Promega (Madison, Wisconsin); the chloramiphenicol acetyltranserase encoding gene is available in the plasmid pCAT-Basic from Promega; the beta-galactosidase encoding gene is available from many commercial sources on many plasmids as the LacZ gene; and a GFP encoding gene is available on plasmid pEGFP from Clontech (Palo Alto, California) . Other sources for these genes are also readily available to one skilled in the art, including the complete sequences of these genes on public sequence databases such as Genbank, and therefore the invention is not to be construed as limited to any particular reporter gene. Insofar as the knockout allele can be present in one or both alleles for N-CAM, it is understood that the invention contemplates a transgenic mouse that is either homozygous or heterozygous for the knockout N-CAM allele, as is described herein.

A preferred transgenic mouse comprises a reporter gene that is the lacZ gene which encodes beta-galactosidase. Preferably, the lacZ insertion into the N-CAM allele has a structure following homologous recombination using plasmid pNCLKO as described in the Examples and as shown in Figure IB.

A beta-galactosidase protein, in the context of the present invention refers to a polypeptide which contains beta- galactosidase activity, and is not intended to be restricted to the native bacterial protein. The LacZ gene has been extensively studied and modified in the art, and it is well known that the enzyme can be presented in a variety of forms and retain the enzyme activity, including truncated proteins, fusion proteins, proteins having modified amino acid residue sequences, and the like.

Thus, in one embodiment, the reporter gene can be a fusion protein having multiple functions. One function is the reporter function, ie, to provide a detectable protein by virtue of an activity or binding property, e.g., as an enzyme, an antigen or the like activity. The second function can be any biological activity to supplement the detection system or to supplement the selection procedure. For example, the fusion protein may include a selectable marker such as imparting resistance in cell culture to cytotoxic agents. a preferred selection marker is the protein which imparts resistance to the neo gene and provides selection resistance to G418 as described in the Examples. Thus, a preferred embodiment is the fusion protein encoded by the LacZ/GPKneo gene present in plasmid pNCLKO described in the Examples . Methods of making disrupted genes according to the present invention, and a transgenic mouse containing such genes are well known to one of ordinary skill in the art. The present invention is based in part on the ability to decrease or completely suppress the level of expression of N-CAM in the mouse by introducing into the genomic DNA of a mouse a new exogenous DNA sequence that serves to interrupt some portion of the endogenous DNA sequence to be suppressed. Another term for this type of suppression is "knockout". Thus, the exogenous nucleic acid is also referred to as a "knockout construct" . Typically, as described in the present invention, the knockout construct, in this case the plasmid or expression vector is first prepared and then inserted into an embryonic stem cell in which the construct subsequently becomes integrated into that cells' genomic DNA by the process of homologous recombination. The embryonic stem cell containing the exogenous DNA is then subjected to selection methods as described herein, for example, by selection for neomycin or G418 resistance. Selected resistant cells are then analyzed for the presence of a mutant allele. An embryonic stem cell containing a mutant allele is then injected into a blastocyst that is implanted into the uterus of a pseudopregnant foster mother for integration into a developing embryo .

Offspring that are born to the foster mother are then screened for the presence of the mutant allele. Typically, a tail sample of the progeny is taken and analyzed by Southern blot hybridization or PCR fragment analysis of genomic DNA using probes specific for the allele, such as is described in the Examples. Animal that contain the N-CAM knockout allele in the germ line are selected and can be used for the generation of homozygous heterozygous animals by sexual crossing. Exemplary teachings of the preparation of disrupted genes and mammals containing such genes are provided in US Patent Nos. 5,553,178, 5,557,032 and 5,569,824, the disclosures of which are hereby incorporated by reference. There are many different genetic backgrounds available for propagating and crossing mice, and therefore the invention is not to be construed as so limited. Methods for identification of a mouse that is heterozygous or homozygous for the knockout N-CAM allele are described herein, and include genotypic verification of the allele by, for example, southern blot hybridization, PCR fragment analysis of genomic DNA, northern blot analysis, and the like, as are well known.

The production of recombinant nucleic acid molecules, oligonucleotides and primers are well established in the art, and are useful methods for the production of the reagents to construct the plasmids, primers and transgenic mice described herein. Oligonucleotides and primers can be chemically synthesized, as is well known, and primers can be used in various recombinant DNA methods such as polymerase chain reaction (PCR) to produce copies of genes of interest, such as are described herein, for use in the plasmids and the mice described herein. B. Cultured Cell Lines Expressing N-CAM/LacZ

The invention further contemplates cultured cells which comprise a reporter gene inserted into a region of an N-CAM gene of a chromosomal N-CAM allele of the cell, such that the region of insertion is located between a transcription start site and a translation start site in said N-CAM gene, and thereby place expression of the reporter gene under the control of the N-CAM promoter .

The cultured cells can be derived from any tissue of a mouse of the present invention, and therefore can be heterozygous or homozygous for the inserted N-CAM allele according to the invention. That is, tissues and/or cells of a transgenic mouse of this invention can be explanted by any of a variety of methods available in the tissue culture arts to establish a culture of cells, and therefore, the invention is not to be construed as so limited.

For example, the explanted cells can be isolated from epithelial or fibroblast tissue, from embryonic, neuronal or endodermal tissue, and the like tissues of the mouse. a preferred tissue is embryonic stem cell tissue, as is described in more detail in the Examples.

In preferred embodiments, a cultured cell comprises a reporter gene as described herein for a transgenic mouse, and more preferably is the lacZ gene. a particularly preferred cultured cell line comprises a reporter gene that comprises the nucleotide sequence of the lacZ gene of plasmid pNCLKO described herein that encodes a beta-galactosidase protein. An exemplary cell is the cultured cell line NCL6 having ATCC Accession No. and described further in the Examples.

The preparation of a cultured cell line can vary widely, and can include the harvesting of a tissue or cell from the mouse, or from a progeny of a mouse, or from a descendant which is the result of a cross which alters the genotype of the mouse, but maintains the knockout allele of the N-CAM gene as described herein.

In one embodiment, it is preferred to engineer a cultured cell so that the cell can be readily propagated in tissue culture, and preferably propagated through multiple generation of cell culture and division of the culture. In ideal culture conditions, the ability to propagate indefinitely is referred to as an immortal cell line, and represents one preferred embodiment of the invention.

There are many ways to immortalize a cultured cell, and therefore, the invention is not to be construed as so limited. a preferred method comprises introducing a gene which provides to the cell the ability to propagate in tissue culture indefinitely. The SV40 virus large T antigen is known to promote the ability to grow in culture, and provides one approach towards immortality. For example, the gene which encodes the SV40 large T antigen is known, and can be introduced into the cell to aid in the establishment of a cell line. In particular, the gene can be introduced before, during or after explanting the tissue from a mouse, although is most convenient to do so prior to explantation. A preferred method involves engineering a mouse strain which contains a genotype which includes a gene capable of expressing SV40 large T antigen under the control of conditional promoters so that expression of the antigen can be turned on during cell culturing, but can be off in the animal. An exemplary genotype is the "Immortomouse" mouse strain produced by Charles River Laboratories (Wilmington, MA) , in which the SV40 large T antigen is under the control of a H-2Kb promoter that is responsive to gamma interferon, and which expresses the antigen as a temperature sensitive (ts) protein that is functional at 33 degrees Centigrade (33 C) , but is nonfunctional ("off") at 39 C. This strain has been described in more detail in the Examples .

A cell line according to this immortalized embodiment is produced by first crossing a mouse having an N-CAM knockout allele with an Immortomouse, screening for progeny containing both genotypes, and explanting tissues from the progeny to form the cultured cell . The genotype screening for appropriate progeny, and the cell culture conditions to establish the cell culture are described in the Examples .

A particularly preferred cultured cell has both the N-CAM knockout allele and the SV40 ts large T antigen, interferon responsive gene. Exemplary is the cell line NCL6 described herein.

C. Methods For Using a Transgenic Mouse or Cultured Cell Line to Screen For Modulators of N-CAM Gene Expression 1. In Vivo Screening of Modulators

The invention contemplates methods for in vivo screening of modulators of the N-CAM gene using a transgenic mouse of the present invention having a reporter (i.e., indicator) gene under the expression control of the N-CAM promoter .

Potential modulators can be administered to the mouse to characterize the in vivo effect of the modulator upon N-CAM expression. Similarly, the mouse can be used to show tissue distribution of N-CAM expression under the influence of a putative modulator to be screened by in situ analysis of expression in different tissues. Alternatively, treatments and/or behavior modifications on the mouse can studied for their effect upon the expression of N-CAM. In addition, the transgenic mouse can be used as tool for screening for biologically active molecules, treatments, behavior modifications or stimuli which modulate N-CAM expression. For example, by administering a test substance to the mouse and observing expression of the reporter gene, the effect of the substance on N-CAM expression can be identified. The substance can be any type of molecule, composition or therapeutic to be evaluated for the ability to modulate N-CAM expression and can comprise a hormone, a small peptide, synthetic analogs, proteins, complex carbohydrates and the like molecules. In a related embodiment, the effect of a treatment, such as a learned or imposed behavior, a physical therapy, a physical stimulus, an electrical or neuro-physiological stimulus or other protocol, when imposed upon the mouse can be evaluated for its effectiveness in modulating N-CAM expression. Combinations of stimuli and chemical substances can also be evaluated.

Thus, the invention describes a method for screening for a bioactive molecule capable of modulating N-CAM expression comprising the steps of: a) contacting a candidate bioactive molecule with a knockout transgenic mouse according to the present invention; and b) evaluating the expression of the reporter gene in a tissue of the mouse, and thereby the ability of the bioactive molecule to effect expression of N-CAM. In practicing the method, the candidate bioactive molecule is typically screened by administration by conventional routes depending upon the class of molecule and the intended therapeutic treatment method. Such methods can include administering the candidate bioactive molecule to the mouse by oral, intravenous, intramuscular, intracranial , subcutaneous, and the like routes.

The detection (ie., evaluating) of the expressed reporter gene can be conducted in a variety of methods, and therefore the invention is not to be construed as so limited. For example, detection can be conducted in situ by detecting the expressed protein in a tissue of the mouse, in vitro by harvesting a tissue and measuring the amount of protein therein, or in vivo by imaging the protein in the whole animal or in a whole organ. Detection is also dependent upon the type of reporter gene. Preferred methods of detection are shown in the Examples.

The use of a transgenic mouse according to the present methods is described in the Examples where ampakine is used to modulate the knockout N-CAM promoter and induce the expression of beta-galactosidase. Other candidate bioactive molecules could be similarly screened in the disclosed methods .

2. In Vitro Screening of Modulators

Cultured cells which contain a reporter gene under the control of the N-CAM promoter can also be used for screening and/or characterizing the effects of potential modulators of the N-CAM promoter.

The cultured cell provides particular advantages over the mouse in terms of the degree of difficulty in conducting the screening method both in terms of costs and the manipulative steps for administration of test compounds and screening for reporter gene expression.

Thus, the invention describes a method for screening for a bioactive molecule capable of modulating N-CAM expression comprising the steps of: a) contacting a candidate bioactive molecule with a cultured mouse cell according to the present invention; and b) evaluating the expression of the reporter gene in the cell, and thereby the ability of the bioactive molecule to effect expression of N-CAM. The contacting step can be conducted by adding compound directly to the tissue culture medium for the cell culture, as is well known, and the cell culture can easily be carried through any culturing conditions as is appropriate for screening. Subsequently, the cells can be treated for evaluation of reporter gene expression. This step can vary widely depending upon the reporter gene and the mechanism for detection of the gene, and can include direct visual inspection following addition of a substrate that reacts with the expressed reporter protein, staining for the expressed protein, immunological detection methods, direct measurement of the protein or protein activity in cell homogenates, and the like methods .

In addition, it is known that a preferred cultured cell, the NCL6 cell, is a pluripotent embryonic cell which can be induced to differentiate into a variety of distinctive differentiated tissues, each of which can provide information regarding regulation of the N-CAM promoter.

D. Therapeutic Methods

The invention further contemplates a therapeutic method comprising administering to a mammal a composition or compound which modulates N-CAM gene expression.

The invention describes an N-CAM modulating compound produced by the screening method of the present invention. In particular, the invention describes a composition that modulates N-CAM expression. Therefore the invention describes a method for increasing N-CAM expression comprising administering to a mammal a composition comprising a therapeutically effective amount of a compound which modulates the N-CAM promoter according to the present screening methods . In preferred embodiments, the composition comprises ampakine. Ampakine is a class of compounds well known in the art, and are allosteric modulators of AMPA receptors that enhance normal glutamate-mediated synaptic transmission. a preferred ampakine is CX547.

Thus the invention describes a method for modulating N-CAM expression, and neuronal activities mediated by N-CAM expression, comprising administering a therapeutically effective amount of a composition comprising ampakine to a patient exhibiting a condition in which modulation of N-CAM expression is beneficial.

The following examples are intended to illustrate, but not limit, the scope of the invention.

Examples In order to examine the potential relationships between N-CAM transcriptional regulation and altered synaptic activity in the adult brain, mice were produced in which the bacterial lacZ gene was inserted into the 3 ' end of the first exon of the N-CAM gene, thereby disrupting N-CAM expression and placing β- galactosidase (β-gal) expression under the control of the N-CAM promoter. The design of this transgenic mouse permitted studies in which N-CAM promoter activity could be assayed conveniently in si tu by measuring β-gal activity in heterozygous animals and correlating the results with parallel measurements of N-CAM mRNA levels (see Fig. 1A) . Mice homozygous for the lacZ insertion lacked N-CAM mRNA and protein and showed many of the phenotypic alterations seen in other N-CAM knockout animals, including decreased migration of cells into the olfactory bulb and displacement of pyramidal cells in the CA3 region of the hippocampus. Tomasiewicz et al , Neuron . 11:1163-1174 (1993); and Cremer et al , Nature, 367:455-459 (1994). However, unlike other N-CAM deficient animals (Muller et al, Neuron , 17:413-422, 1996), they exhibited normal hippocampal LTP.

1. Preparation of a Plasmid Vector Construct For Producing a Knock-Out Gene Based On N-CAM/LacZ a targeted replacement vector was designed to disrupt the N-CAM allele and to insert a lacZ reporter gene under control of the endogenous N-CAM regulatory sequences (Figure IB) . The vector contained the 8 kb XhoI-SacII fragment of the N-CAM promoter and the 2 kb Notl-EcoRI fragment as 5¹ and 3' homologous recombinant arms, respectively. The region between the SacII and Notl sites, which includes most of exon 1 and a small part of the first intron, was replaced by an E. coli lacZ gene cassette containing an ATG codon followed by a nuclear localization signal and the neomycin resistance gene driven by the phosphoglycerate kinase (PGK) promoter. Adra et al, Gene , 60:65-74 (1987). The lacZ gene was isolated as an Xbal-Hindlll fragment from placF, has a known nucleotide sequence (GenBank accession number V00296) that encodes β-galactosidase, an enzyme whose activity is readily detected and is not toxic. To allow for selection against random insertion, a PGK-thyτnidine kinase gene was inserted at the 3 ' end of the N-CAM homologous sequences .

The N-CAM genes were obtained from an N-CAM genomic clone which was isolated from the mouse 129/Sv genomic library

(Clonetech) and compared with previously defined N-CAM gene structure. Hirsch et al , Mol . Cell .Biol .. 10:1959-1968 (1990): and Jones et al , Proc . Natl .Acad . Sci . USA. 89:2086-2090 (1992). The probes for the screen of the 129/Sv library were a BamHi-Pst fragment from pEC9.7 according to Jones et al, ibid. Several clones were identified which were used to generate an 8 kb Xhol- SacII fragment of the N-CAM promoter which was used as the 5' homologous recombinant arm and a 2 kb Notl-EcoRI fragment which was used as the 3 ' homologous recombinant arm of the construct in plasmid pNCLKO (Figure IB) . Figure IB shows the structures of the N-CAM targeting vector, wild-type N-CAM allele, and the disrupted N-CAM allele that was generated after homologous recombination. Restriction enzyme sites are abbreviated as: E, EcoRI ; K, Kpnl; N, NotI; S, Sail; Sa, SacII; X, Xhol. Hatched boxes above the diagram of disrupted N-CAM allele indicate restriction fragments used as probes for Southern blot analysis of the ES cell lines. Probe a is made from the BamHI-Pstl promoter fragment and probe b is made from the Kpnl -Xhol intron 1 fragment of pEC9.7.

Using the probes in Southern blots, the following sizes of fragments are obtained which were detected for the wild type or disrupted N-CAM alleles probed with the indicated probes and restriction enzymes:

Probe Restriction Fragment Fragment Detected (kb)

Wild Type Disrupted a Sail + Xhol 14 9 b Sail + Xhol 14 7 b NotI + Xhol 5 10.5

The resulting plasmid, having the features shown in Figure IB, was designated as plasmid pNCLKO, and has been deposited with the ATCC as described herein.

The following restriction digests fragments are diagnostic for the plasmid pNCLKO: Restriction Enzyme Nucleic Acid Fragment Size Xho I 21 kb (linear)

Sal I 17, 3.6 kb

Not I 11, 7.6, 2.7 kb Kpn I + Sac II 9, 5.6, 4.7, 3 kb.

Other similar probes can also be prepared according to the restriction maps shown in Figure 1 and using plasmid pNCLKO and the published sequence information on the N-CAM allele to monitor the progress of the homologous recombination process.

2. Construction of Transgenic N-CAM Knock-Out Mouse a transgenic N-CAM knockout mouse was produced having the construct present in plasmid pNCLKO in which the native N-CAM allele is disrupted by homologous recombination to produce a substituted allele which contains the LacZ gene under the expression control the N-CAM promoter.

The site of the homologous recombination was between the transcriptional and translational start sites. The E.coli lacZ gene encoding the beta-galactosidase was inserted in the native N-CAM gene (Figure IB) . Homologous recombination in the 129/Sv ES cell line resulted in a mutant allele with the lacZ reporter gene under control of the transcriptional regulatory regions of the N-CAM gene while at the same time blocking expression of the wild type N-CAM protein.

The plasmid pNCLKO was introduced into the embryonic stem cell strain ES-D3 using electroporation to form a transfected cell. To that end, 50 ug of plasmid DNA was electroporated onto 7.5 x 10⁷ ES-D3 cells, and selection was conducted using G418. The colony forming frequency following electroporation and selection was 5.2 x 10^~6 colonies per cell. Twenty five colonies were isolated and were implanted into blastocysts generated from a C57B1/6J mouse for producing a transgenic mouse.

ES-D3 cells are an established stem cell culture prepared from pluripotent stem cells of an mouse 129 strain which spontaneously differentiate into embryonic structures in culture. ES-D3 cells are available from ATCC as Accession No. CRL-11632.

Of 16 transfected 129/SV ES cell clones (from ES cell clone D) selected for G418 resistance, five contained the recombined allele as assessed by Southern blot analysis of Notl/Xhol digests of genomic DNA probed with probe a (Figure IB; data not shown), including the 10.5 kb fragment indicating homologous recombination to form the disrupted allele. Cells were subjected to karyotype analysis to insure a normal chromosome complement. a single ES cell clone with the identified disrupted allele and a normal karyotype was injected into C57B1/6J blastocysts to generate chimeric founder mice. One of the chimeric animals transmitted the recombinant allele through the germline resulting in animals heterozygous for the N-CAM null allele. The offspring of this animal were used to establish heterozygous and homozygous animals in the background of the inbred C57B1/6J mouse strain and the outbred CD-I strain.

3. Methods for Genotype Verification of a Knock-Out Mouse PCR analysis of mouse tail DNA was used to genotype progeny, and was performed as described by Wang et al , Proc . Nat1.Acad . Sci . USA. 93:1892-1896 (1996). To identify the recombined allele a PCR fragment of 305 base pairs (bp) is detected and to identify the endogenous wild type (wt) allele a PCR fragment of 425 bp is detected, each using primers designed as described below.

To detect the endogenous N-CAM allele, we sequenced the first 350 base pairs (bp) of the first intron of the N-CAM gene, and the sequence obtained was used to design a reverse 3 ' primer (5' ATG GCT CCC TTC TCA GCT CAG TG 3 ' ) (SEQ ID NO 1) . This reverse 3' primer was used in combination with a 5' primer (5' ATT CTC CGC TCA GCG CGT GAA C 3') (SEQ ID NO 2) that is complementary to the -168 to -189 region of the N-CAM promoter to amplify a 425 bp PCR fragment. To detect the disrupted N-CAM allele containing the lacZ gene, a 3' primer was designed (5' CGC CAG GGT TTT CCC AGT CAC GAC G 3') (SEQ ID NO 3) that corresponds to the M13 (-40) sequencing primer located near the polylinker region of the pnLacF vector. This M13 3' primer was used in combination with the above 5 ' primer complementary to the N-CAM promoter to amplify a 305 bp PCR fragment.

The expected PCR products from the recombined allele (305 bp) and from the endogenous allele (425 bp) were visualized on an ethidium bromide-stained agarose gel after electrophoresis . Animals with tail DNA samples that contain both products (305 bp and 425 bp) indicate an animal heterozygous for the disrupted allele, ie, has one wild-type and one knock-out allele for the N-CAM gene (+/-) .

4. Phenotype Studies of N-CAM/LacZ Knock-Out Mouse

The phenotype of the N-CAM/lacZ knock-out mouse was verified by both northern blot analysis and western blot analysis. Mice homozygous for the disrupted N-CAM allele had no expression of either N-CAM protein (western) or N-CAM mRNA (northern) . a . Northern Blots

Northern blot analysis was conducted on the knockout mouse to characterize the expression of mRNA corresponding to the gene for the N-CAM/LacZ construct, and to verify the expression of the wild type allele for N-CAM in wild-type mice or mice heterozygous for N-CAM/LacZ. In Northern blot analysis (Fig. IC) , a probe corresponding to base pairs 183 to 505 of the N-CAM cDNA (according to numbering in GenBank accession number X15049) hybridized to RNA from wild type and heterozygous mouse brains but not to RNA from mice homozygous for the N-CAM gene disruption. Ethidium bromide staining indicated that similar amounts of RNA were loaded on the gel (Fig. ID) .

b . Western Blots Western blot analysis was conducted on extracts of brain tissue to characterize the expression of N-CAM proteins using a polyclonal anti-N-CAM antibody. The polyclonal antibody was produced by conventional immunization of isolated mouse N- CAM protein into rabbit, and harvesting of the sera. Suitable anti-N-CAM antibody, also referred to as "anti-CD56", can also be obtained from a variety of commercial sources, such as Becton Dickinson (Franklin Lakes, New Jersey) .

In Western blot analysis of total brain proteins, a polyclonal antibody to N-CAM recognized three protein bands corresponding to the major 120, 140 and 180-kDa isoforms of

N-CAM in the extracts from the wild type and heterozygous mice (Fig. IE) . The N-CAM antibody did not bind to any proteins in the extracts from mice homozygous for the N-CAM gene disruption. Insertion of the bacterial lacZ gene into the first exon and intron of the N-CAM gene disrupted expression of both N-CAM mRNA and protein (Figure IC, E) . Mice homozygous for the gene disruption expressed no gross phenotypic abnormalities other than a reduced body size and decreased breeding efficiency. As detailed below, the knockout mice described herein have some of the morphological defects observed in other mutants (Tomasiewicz et al, Neuron, 11:1163-1174 (1993); and Cremer et al , Nature, 367:455-459, 1994) although the various N-CAM knockout strains described here and by others represent different alleles due to mutation at widely distributed sites within the N-CAM gene.

c . Beta-Galactosidase Expression

1. β-gal histological staining

Whole mount staining of embryos was performed as previously described. Wang et al, Proc .Natl .Acad. Sci .USA. 93:1892-1896 (1996); and Hoist et al, Proc . Natl . Acad . Sci . USA. 94:1465-1470 (1997) . Ampakine- or vehicle-treated animals were fixed by perfusion with 1% formaldehyde/0.25% glutaraldehyde in PBS. Brains were removed and postfixed for 1 h at 4°C, infiltrated with sucrose, and 50 μm cryosections were processed as described by Wang et al . , ibid. By titrating the time of postfixation, we were able empirically to determine conditions that minimized activity in control animals and allowed observation of differences in β-gal staining.

2. β-gal enzymatic assay Two to three millimeter sagittal sections of the hippocampus from each hemisphere were isolated and prepared individually. The tissue was lysed by sonication in 100 mM Tris-acetate, pH 7.8 , 10 mM Mg acetate, 1 mM EDTA, 1% TX-100, 0.2% deoxycholate . Lysate was assayed for β-gal activity with the FluoReporter kit (Molecular Probes, Eugene, Oregon) according to manufacturer's protocols. β-gal values were normalized to DNA content of the samples as determined by the Picogreen assay (Molecular Probes) . The values for β-gal from each hemisphere were averaged. Within an experimental group, β-gal activity for individual treatments was compared to the average β-gal levels for control animals. The percent increase was averaged over all experiments and analyzed statistically using the Wilcoxon matched pairs test.

3. Results Inserting the bacterial lacZ gene into the N-CAM gene placed β-gal expression under the regulation of the native N-CAM promoter. To establish that β-gal expression was regulated appropriately by the N-CAM promoter, β-gal expression patterns were examined using enzymatic assays in heterozygous and homozygous embryos and compared them to N-CAM mRNA expression patterns as assessed by in si tu hybridization. In E9.5 and 13.5 embryos, β-gal staining was observed in a pattern similar to that observed for N-CAM mRNA (Fig. 2) and was seen only in tissues which express N-CAM mRNA. At E9.5, β-gal staining was expressed prominently throughout the spinal cord and in the dorsal root ganglia (Fig. 2A, B) . By E13.5, β-gal continued to be expressed throughout the spinal cord and in the dorsal root ganglia (Fig 2C, D) and in heart and kidney (data not shown) and was similar to the pattern of in si tu hybridization for N-CAM mRNA (Fig. 2F) . At this stage, there was increased β-gal activity in the brain, particularly in the area of the hindbrain, midbrain, and the floor plate of the forebrain as well as in the trigeminal ganglia (Fig. 2G) . In the head, β-gal expression was increased and was localized to post-mitotic neurons of the midbrain and hindbrain and along the floor plate of the forebrain (Fig. 2G) comparable to the pattern of N-CAM mRNA expression (Fig. 2H) . The β-gal staining patterns demonstrated that β-gal expression was regulated by the N-CAM promoter in both homozygous and heterozygous mice . These data indicate that β-gal is expressed in a pattern similar to that of native N-CAM mRNA. These findings also indicate that β-gal expression serves in these mice as a valid reporter of N-CAM expression.

d. Hippocampal morphology in N-CAM deficient mice Homozygous knockout mice had a bifurcation of the CA3 region of the hippocampus not present in wild type animals (Fig. 3A, B) , but similar to that reported in other N-CAM knockout mice (Tomasiewicz et al , Neuron . 11:1163-1174 (1993); and Cremer et al, Mol . Cell. Neurosci.. 8:323-335, 1997). In addition, the homozygous mice appeared to have fewer pyramidal cell nuclei in the CA1, CA2 and CA3 regions of the hippocampus. Heterozygous animals showed an intermediate morphological phenotype with more pyramidal neurons and a less pronounced bifurcation. In spite of these defects, the projections from the dentate gyrus to the CA3 region were similar in mutant and wild type animals as revealed by Timm's staining. It was also apparent in these sections that homozygous animals had an increase in the number of cells in the sub-ventricular zone on the route of migration to the olfactory bulb. They also showed a reduction in the size of the olfactory bulb.

e . Electrophysiology in N-CAM Deficient Mice Transverse slices of hippocampus (450 μm) were prepared from 12 to 20 week old mice by parasagittal section as previously described for rats (Langdon et al , J. Physiol ..

472:157-176, 1993); all operations were conducted in artificial cerebrospinal fluid (ACSF) . During recordings, slices were bathed on all sides by freely flowing ACSF at 32 to 33 °C. Stimulation was provided via fine stainless steel wire electrodes (25 to 50 μm in diameter) placed approximately 300 μm to each side of a glass micropipette recording electrode (ACSF filled; 2-5 MΩ) in the stratum radiatum (Fig. 2C) . Test stimuli were applied at 0.1 Hz, alternating between sites (ie., 0.05 Hz per site) . The stimulus-driven field excitatory post-synaptic potentials (fEPSPs) were amplified with an AxoClamp 2B amplifier to a final gain of 1000, low-pass filtered at 2 or 5 kHz, digitized at 5 or 10 kHz, analyzed on-line and then stored on disk for further analysis.

Recording from each slice began with examination of its input-output relationship. In all cases, it was found that a fEPSP of 2 to 4 mV could be elicited with a stimulus current no greater than 80 μA for 200 μsec . Subsequently, for the LTP experiments, the applied stimulation level was adjusted to evoke fEPSPs of 40 to 60% of maximal amplitude. After at least 15 min of baseline observation, theta burst stimulation (TBS) was applied to the orthodromic pathway. This consisted of nested trains of 4 stimuli at 100 Hz, repeated 6 times at 5 Hz, and this repeated twice at 0.1 Hz. The stimulus current amplitude and duration were the same during conditioning as during test cycling. The data from an LTP run were considered acceptable if responses to the non-conditioned input deviated in amplitude by less than 20% of initial value. The LTP group statistics were derived from all such runs, regardless of the magnitude of LTP observed. Group statistics were derived after calculating an averaged outcome for each animal; hence, the stated sample sizes are animal counts, rather than the actual number of slices tested (2 to 3 per animal) . Baseline synaptic physiology and LTP was measured in hippocampal slices from heterozygous and homozygous N-CAM knockout mice in comparison with those of wild type animals . Synaptic physiology was studied in the Schaffer collaterals in hippocampal slices prepared from 7 homozygous mutant mice (4 from the CD1 background, 3 from the C57B1/6 background) , 2 heterozygotes, and 6 wild type animals (Fig. 3C) . The field excitatory postsynaptic potentials (fEPSPs) measured in these groups were indistinguishable with respect to size and shape, and were typical of those reported in the literature for slices from normal rodents. The fEPSP began after a latency of 2.5 to 3 msec, rose in 1.5 to 2.5 msec, and decayed with a half-width of about 8 msec (Table 1) . Paired-pulse facilitation of the fEPSPs was compared in slices from the homozygous mutant and wild type mice, using an interstimulus interval of 25 ms (Table 1 and Fig. 3D) . Mean facilitation ratios of 1.60 + 0.06 and 1.47 ± 0.03 were observed, which did not differ significantly and were similar to levels described in previous studies of these synapses. Half-maximal baseline fEPSPs used for LTP runs were 1 to 2 mV and were elicited by stimulus currents between 20 and 25 μA.

TABLE 1:

Conditions and fEPSP parameters in synaptic physiology experiments

Presynaptic

Stimulus volley, peak current, amplitude, fEPSP peak Genotype μ X 0.2ms mV amplitude. V n

-/- 24.9 ± 3.1 0.13 ± 0.02 1.27 ± 0.09 7

-/+ 24.7 ± 3.7 0.19 + 0.01 1.08 ± 0.10 2

+/+ 24.3 ± 3.4 0.17 ± 0.01 1.56 ± 0.14 6 fEPSP fEPSP latency to risetime (10 fEPSP

Genotype 10% rise, ms to 90%) . ms halfwidth. ms n

-/- 2.97 ± 0.11 2.23 ± 0, .09 7.90 ± 0.29 7

-/+ 2.09 ± 0.20 1.68 ± 0, .03 8.01 ± 0.01 2

+/+ 2.87 ± 0.29 2.20 ± 0, .15 7.72 ± 0.35 6

paired-pulse facilitation

Genotype ratio n

-/- 1.60 ± 0.06 7

-/+ n.d. 2

+/+ 1.47 ± 0.03 6

Data is expressed as a group means + SEM.

The slices were also examined with respect to theta burst stimulation (TBS) -induced LTP. Robust LTP, found in slices from all groups, was stable for the duration of the recordings regardless of whether N-CAM was expressed or not (Fig. 3F, G) . Our findings were essentially the same whether response amplitudes were determined at fEPSP peak or initial slope (Fig. 3F, G) . Typically, TBS in homozygous mutant and wild type animals elicited a brief (< 2 min) post-tetanic potentiation (PTP) , a variable and also brief (< 5 min) heterosynaptic depression, short-term potentiation, and LTP, the latter two phenomena being superimposed for the first 20 to 30 min following TBS. As determined from the initial slope of the fEPSP before applying TBS and 45 min after, the mean LTP magnitude (in percent increase) was 64.7 + 10.9, and 56.9 +_. 9.9 in slices from the homozygous mutant and wild type animals, respectively. The means did not differ significantly when tested at the level of p < 0.05 (t test) . Comparable short- and long-term plasticity was observed in slices prepared from heterozygous mutants. In summary, mice that lacked N-CAM exhibited normal LTP and baseline synaptic physiology. f . Ampakine Effects in N-CAM Deficient Mice

Heterozygous mice were used to examine regulation of the N-CAM promoter in response to enhanced synaptic transmission since, in these mice, β-gal expression from the lacZ gene insertion and N-CAM mRNA expression could both be used to monitor the activity of the N-CAM promoter. Synaptic activity was enhanced by treatment with ampakines, a class of drugs that are positive allosteric modulators of AMPA receptors which increase the amplitude and prolong the time course of synaptic responses to endogenous glutamate release. To assay the response of the N-CAM promoter to increased synaptic transmission in vivo and in vi tro, the ampakine CX547, a positive allosteric modulator of alpha-amino-3 -hydroxy-5-methyl-4-isoazole propionic acid (AMPA) -type glutamate receptors, was used.

Heterozygous mice were injected intraperitoneally (i.p.) with 80 mg/kg of the ampakine CX547 dissolved in 50% PBS with 20% w/v of the carrier cyclodextran. Control animals were injected i.p. with the vehicle alone (20% cyclodextran w/v in 50% PBS) .

Eight hours after treatment with ampakine, the brains were harvested, sectioned, and stained histologically for β-gal activity. Hippocampal tissue was also homogenized and assayed for β-gal activity as described earlier. After homogenization of individual brains, N-CAM mRNA levels were also measured by RNAse protection as described below.

Histological examination of sagittal sections of the hippocampus was carried out as described above in Example 4. c .1 and revealed an increase in the level of b-gal expression driven by the N-CAM promoter (Fig. 4) . When examined histologically, β-gal staining increased after 8 h in heterozygous mice. The most dramatic changes were in the CA1 and CA2 regions of the hippocampus where a marked increase in the intensity of staining was observed relative to control mice treated with vehicle alone (Fig. 4) . Increases in β-gal expression were also observed in areas outside of the hippocampus, most notably in the deep layers of the cortex (Fig. 4) . At this level of analysis, however, β-gal staining did not appear to change in the superficial layers of the cortex.

To quantitate the percentage increase in N-CAM promoter activation in response to ampakine that was observed histologically, β-gal assays were performed on extracts of hippocampus isolated from mice 8 h after these animals had been treated with either CX547 or carrier. An average increase of 19% in β-gal activity was observed in the hippocampus as compared to that of vehicle-injected animals (Table 2) . The data indicate that ampakine administration increases N-CAM promoter activity in vivo .

TABLE 2 :

Percent changes in β-gal activity and N-CAM mRNA following ampakine treatment

in vi vo Mean treatments 1 2 3 4 5 6 7 % increase β-gal 25 38 13, 33 1, 17, 20, 19+3* hippocampus) , 2 29, 38, 12,

% increase 9 10 22

m vi vo Mean treatments 1 2 3 4 5 6 7 % increase

N-CAM mRNA ND ND 28 63 21, 28, 8, 26±6**

(total brain 32, 46, -13,

RNA) , 47 3 22

% increase in vi tro Mean treatments 1 2 3 4 5. % increase β-gal 58, 59, -40, 77 22, 27₊8*** hippocampal 31, 3, 31, 41, 8 slices) , 24, 0 54, 16

% increase 26 -2

in vi tro Mean % treatments 1 2 3 4 5 suppression β-gal ND ND ND 42 100, 89+22****

% suppression 35, 150 by CNQX of 118 ampakine induction

Changes in β-gal activity and mRNA are expressed as percentages over or under control values . For average percent increase, values are group means ± SEM. Statistics in Wilcoxon matched pairs test: *p=<0.001; **p=<0.003 ****Students t-test, p=<0.004.

Animals were killed 8h following i.p. injection of 80mg/kg of ampakine. β-gal assays were performed on isolated hipppocampi and RPAs for N-CAM RNA were performed on total brain RNA. N.D. = not done.

To obtain an independent confirmation of the degree of ampakine stimulation of N-CAM promoter activity, we performed RNAse protection assays (RPAs) on total RNA isolated from the brain tissue of the heterozygous mice that had been assayed for β-gal activity following ampakine treatment.

RNAse protection assays were performed on total brain RNA isolated using RNAzol (Tel-Test, Friendswood, TX) extraction from ampakine- or vehicle-treated animals. A ³²P-labeled probe for the region of the N-CAM mRNA (ntl83 to 505 according to Genbank number X15049) and β-actin (Ambion, Austin, Texas) was used in the Ambion RNAse protection assay according to manufacturer's protocols. Protected fragments were visualized and quantitated on a phosphoimager (Molecular Dynamics, Sunnyvale, California) . N-CAM values were normalized to levels of β-actin mRNA as determined by the protection assay. Values were analyzed statistically by the Wilcoxon matched pairs test. Using a probe that hybridizes to all isoforms of N-CAM mRNA in RPA, we observed an average increase of 26% in N-CAM mRNA expression of in the ampakine-treated mice over control animals (Table 2) . These data indicate that, in the heterozygous animals, ampakine treatment results in increases in levels of endogenous N-CAM mRNA of the same magnitude as those of β-gal expression driven by the N-CAM promoter.

To assay the effect of heightened glutamatergic transmission on N-CAM promoter activity in a system which is more amenable to experimental manipulation, hippocampal slices were cultured from mice heterozygous for the lacZ insertion into the N-CAM gene and the slices were then exposed to ampakines in vi tro .

To that end hippocampal slices were prepared for culture from heterozygous mice as described previously (Vanderklish et al, Mol. Brain Res.. 32:25-35, 1995). After 7 to 10 days in culture, slices were incubated in 200 μM CX547 or carrier for 30 min. after which the medium was exchanged and the slices incubated for an additional 7.5 h. Slices were harvested in a buffer composed of 0.32M sucrose, 2mM EDTA, 2mM EGTA, 25mM HEPES, 50 μM leupeptin, pH 7.4 on ice, collected by centrifugation, and lysed by sonication in the β-gal assay buffer as described above.

Following incubation of slices in 200 μM CX547 for 30 min, an average increase of 27% in β-gal activity was found in the hippocampal slices at 8 h posttreatment (Table 2) . An increase of 20% was also observed in the presence of the AMPA receptor agonist, kainic acid. The response to ampakine was strongly reduced in the presence of the AMPA receptor antagonist CNQX (average decrease = 89 % ± 22) , indicating that activation of the N-CAM promoter was dependent on the ability of the ampakine to influence synaptic transmission through AMPA receptors (Table 2) . This conclusion was further supported by the finding, in control experiments, that embryonic fibroblasts prepared from the heterozygous embryos showed no significant changes in N-CAM promoter activity as assessed by β-gal expression when treated with either ampakine or CNQX. These data indicate that ampakine treatment increases N-CAM promoter activity in organotypic slice cultures in vi tro .

g. Discussion The above studies demonstrate that mice expressing lacZ under the control of the N-CAM promoter showed increased β-gal reporter activity and N-CAM mRNA levels in response to ampakine treatment. By utilizing mice heterozygous for the lacZ insertion, it is demonstrated in the same animal that there were similar increases in the levels of both N-CAM mRNA and β-gal activity. In control experiments, CX547 had no effect on fibroblasts cultured from heterozygous embryos and the effects of the drug on tissue slices was reduced by CNQX, a specific antagonist of AMPA receptors. These findings indicate that enhanced synaptic transmission leads to activation of the N-CAM promoter and that β-gal expression in these mice can be used in further studies as an indicator of patterns of neural activity in in vivo .

After ampakine treatment in heterozygous mice, an increase in N-CAM promoter activity was observed as assessed by the β-gal reporter. N-CAM mRNA increased in ampakine-treated heterozygous knockout mice and wild type mice as shown by RNAse protection assays (RPA) . The response to ampakine was also observed in organotypic slice cultures of the hippocampus. In such slices, the response was similar to that found when glutamate receptors were stimulated with the agonist kainic acid and it was prevented by CNQX, an AMPA receptor antagonist. Together these results indicate that facilitation of AMPA receptors leads to increased N-CAM promoter activity.

Treatment of the mice with an ampakine, an allosteric modulator of AMPA receptors that enhances normal glutamate-mediated synaptic transmission, increased the expression of β-galactosidase in vivo as well as in tissue slices in vi tro . Similar treatments also increased the expression of N-CAM mRNA in the heterozygotes . The effects of ampakine in slices were strongly reduced in the presence of CNQX, an AMPA receptor antagonist. Taken together, these results indicate that facilitation of AMPA receptor-mediated transmission leads to activation of the N-CAM promoter and provide support for the hypothesis that N-CAM synthesis is regulated in part by synaptic activity.

5. Preparation of Cell Culture Containing the N-CAM/LacZ Knockout Gene

An established cultured cell line which expresses LacZ under the control of the N-Cam promoter is prepared by crossing in the first generation a male homozygous N-CAM knockout transgenic mouse with a female heterozygous Immortomouse (Charles River Laboratories, Wilmington, MA). The construction and characterization of Immortomouse is described by Jat et al . , Proc . Natl . Acad . Sci . USA. 88:5096-5100 (1991).

Immortomouse is a transgenic mouse which contains a gene that expresses the SV-40 large T antigen with a temperature sensitive tsA58 mutation under the control of a gamma interferon-inducible H-2Kb promoter.

Male and female progeny containing both a heterozygous Immortomouse allele and a heterozygous N-CAM knockout allele were selected and crossed again. Day 17-18 embryos from the second mating were used to create individual hippocampal stem cell cultures.

Stem cell cultures were grown at 33 C in Neurobasal media, GibcoBRL cat. No. 21103-049 supplemented with the following additives (final concentrations noted in parentheses) : B27 supplement, GibcoBRL cat. No. 17504-044, (10 ml per 500 ml of neurobasal media); penicillin, GibcoBRL cat. No. 15070-063, (50 U/ml); streptomycin sulfate, GibcoBRL cat. No. 15070-063, (50 ng/ml) ; L-glutamine, GibcoBRL cat. No. 25030-081, (2 mM) ; gamma interferon, GibcoBRL cat. No. 13283-015, (100 U/ml); and bFGF, Sigma cat. No. F0291, (20 ng/ml)

PCR analysis of genomic DNA isolated from tail samples from the pups was used to identify those cultures made from pups which were heterozygous for the immortomouse gene and either homozygous, heterozygous or wild type for the N-CAM knockout allele .

Verification of the Immortomouse allele in progeny is accomplished using PCR of genomic DNA isolated from the tail samples using PCR primers specific for the Immortomouse' s large T antigen expression construct. The primers used are 5 ' -GCA GAA CTA AGA AGT CGC GA-3 ' (SEQ ID NO 4) and 5 ' -GAC ACT CTA TGC CTG TGT GG-3 ' (SEQ ID NO 5), which hybridize to the H-2Kb promoter domain of the large T antigen construct and to the structural protein for the large T antigen. If the Immortomouse allele is present, the PCR fragment is approximately 1000 base pairs. The protocol for genotyping the Immortomouse uses the two primer in a single reaction to detect a specific PCR fragment. The PCR reaction mix contains 400 nanograms (ng) genomic DNA, 2.5 ul DMSO (5% of total reaction volume), 1 ul 10 mM dNTP, 0.25 ul Taq Polymerase (1.25 units), 5 ul 10xMgCl2 Taq Polymerase buffer, 200 ng of each primer, sterile water for a total reaction volume of 50 ul . The PCR reaction was run once at 95 C for 3 min, 30 cycles as follows: 95 C for 1 min, 62 C for 1 min, and 72 C for 1 min; followed by one cycle at 72 C for 8 min. Stem cell culture produced by a heterozygous N-CAM knockout mouse identified by the above PCR genotype analysis was selected and designated as stem cell culture NCL6. NCL6 was deposited with the ATCC as described herein.

6. Deposit of Materials

The following materials have been deposited on February 19, 1999, with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Virginia, USA:

Deposit ATCC Accession No.

Plasmid pNCLKO 203784

Cell Line NCL6

The deposits listed above, pNCLKO and NCL6 are prepared as described in the Examples . These deposits were made with the ATCC under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) . This assures maintenance of a viable culture for 30 years from the date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty which assures permanent and unrestricted availability of the progeny of the culture to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638) . The assignee of the present application has agreed that if the culture deposit should die or be lost or destroyed when cultivated under suitable conditions, it will be promptly replaced on notification with a viable specimen of the same culture. Availability of the deposited strain is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the cell lines deposited, since the deposited embodiment is intended as a single illustration of one aspect of the invention and any cell lines that are functionally equivalent are within the scope of this invention. The deposit of material does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustration that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims .

Claims

What Is Claimed Is:

1. A transgenic mouse having a reporter gene inserted into a region of an N-CAM gene of a chromosomal N-CAM allele of said mouse, wherein said region is located between a transcription start site and a translation start site in said N-CAM gene.

2. The transgenic mouse of claim 1 wherein said insertion is homozygous .

3. The transgenic mouse of claim 1 wherein said insertion is heterozygous .

4. The transgenic mouse of claim 1 wherein said reporter gene is the bacterial lacZ gene that encodes beta-galactosidase.

5. The transgenic mouse of claim 4 wherein said inserted lacZ gene comprises a nucleotide sequence of plasmid pNCLKO having ATCC Accession No. 203784 that encodes a beta- galactosidase protein.

6. A cultured mouse cell derived from the transgenic mouse of claim 1.

7. A cultured mouse cell having a reporter gene inserted into a region of an N-CAM gene of a chromosomal N-CAM allele of said mouse cell, wherein said region is located between a transcription start site and a translation start site in said N- CAM gene .

8. The cultured mouse cell of claim 7 wherein said insertion is homozygous .

9. The cultured mouse cell of claim 7 wherein said insertion is heterozygous .

10. The cultured mouse cell of claim 7 wherein said reporter gene is the bacterial lacZ gene that encodes beta-galactosidase.

11. The cultured mouse cell of claim 10 wherein said inserted lacZ gene comprises a nucleotide sequence of plasmid pNCLKO having ATCC Accession No. 203784 that encodes a beta- galactosidase protein.

12 . The cultured mouse cell of claim 7 wherein said cell is an immortalized cell line which expresses the SV40 large T antigen.

13. The cultured mouse cell of claim 7 wherein said cell is an embryonic stem cell.

14. The culture mouse cell of claim 10 wherein said cell is the cell line NCL6 having ATCC Accession No. .

15. A method for screening for a bioactive molecule capable of modulating N-CAM expression comprising the steps of: a) contacting a candidate bioactive molecule with a knockout transgenic mouse having a reporter gene inserted into a region of an N-CAM gene of a chromosomal N-CAM allele of said mouse, wherein said region is located between a transcription start site and a translation start site in said N-CAM gene; and b) evaluating the expression of said reporter gene in a tissue of said mouse, and thereby the ability of said bioactive molecule to effect expression of N-CAM.

16. The method of claim 15 wherein said contacting comprises administering said candidate bioactive molecule to said mouse by oral, intravenous, intramuscular, intracranial and subcutaneous routes .

17. The method of claim 15 wherein said reporter gene is the bacterial lacZ gene that encodes beta-galactosidase.

18. The method of claim 15 wherein said inserted lacZ gene comprises a nucleotide sequence of plasmid pNCLKO having ATCC

Accession No. 203784 that encodes a beta-galactosidase protein.

19. The method of claim 15 wherein said evaluating comprises measuring beta-galactosidase activity in said mouse tissues.

20. The method of claim 15 wherein said evaluating comprises histological staining of a tissue section for beta-galactosidase activity.

21. A method for screening for a bioactive molecule capable of modulating N-CAM expression comprising the steps of: a) contacting a candidate bioactive molecule with a cultured mouse cell having a reporter gene inserted into a region of an N-CAM gene of a chromosomal N-CAM allele of said mouse, wherein said region is located between a transcription start site and a translation start site in said N-CAM gene; and b) evaluating the expression of said reporter gene in said cell, and thereby the ability of said bioactive molecule to effect expression of N-CAM.

22. The method of claim 21 wherein said reporter gene is the bacterial lacZ gene that encodes beta-galactosidase.

23. The method of claim 22 wherein said inserted lacZ gene comprises a nucleotide sequence of plasmid pNCLKO having ATCC

Accession No. 203784 that encodes a beta-galactosidase protein.

24. The method of claim 22 wherein said evaluating comprises measuring beta-galactosidase activity in said cell.

25. The method of claim 22 wherein said evaluating comprises histological staining of said cell for beta-galactosidase activity.

26. The method of claim 22 wherein said cell is the cell line NCL6 having ATCC Accession No. .

27. A composition for modulation of N-CAM expression comprising a therapeutically effective amount of an ampakine.

28. The composition of claim 27 wherein said ampakine is ampakine CX547.