Patent Application
20070074297
This application claims the benefit of European Application No. 05108145.3, field Sep. 6, 2005, which is hereby incorporated by reference in its entirety.
Trace amines (TAs) are endogenous compounds related to biogenic amine neurotransmitters and present in the mammalian nervous system in trace amounts. The intense research efforts on the pharmacology and metabolism of trace amines during the last decades has been triggered by their tight link to a variety of highly prevalent conditions such as depression, schizophrenia, anxiety disorders, bipolar disorder, attention deficit hyperactivity disorder, neurological diseases such as Parkinson's Disease, epilepsy, migraine, hypertension, substance abuse and metabolic disorders such as eating disorders, diabetes, obesity and dyslipidemia (Reviewed in: Lindemann & Höner, Trends Pharmacol Sci. 2005; 26(5):274-81 Branchek, T. A. and Blackburn, T. P. (2003) Curr. Opin. Pharmacol. 3, 90-97; Premont, R. T., Gainetdinov, R. R., Caron, M. G. (2001) Proc. Natl. Acad. Sci. 98, 9474-9475.; Davenport, A. P. (2003) Curr. Opin. Pharmacol. 3,127-134.). However, a detailed understanding of the physiology of trace amines on the molecular level has only become possible with the recent identification of a novel family of G protein-coupled receptors termed Trace Amine Associated Receptors (TAARs; Lindemann, L., Ebeling, M., Kratochwil, N. A., Bunzow J. R., Grandy, D. K., Hoener, M. C. (2005) Genomics 85, 372-385; Bunzow, et al. (2001) Mol. Pharmacol. 60,1181-1188; Borowsky, B., et al., (2001). Proc. Natl. Acad. Sci. U.S.A. 98, 8966-8971). Some of these receptors display sensitivity to trace amines, and their unique pharmacology and expression pattern make these receptors prime candidates for targets in drug development in the context of several diseases, some of which previously had been linked to trace amines. Progress in understanding the physiological relevance of Trace Amine Associated Receptors and their ligands on the systems level critically depends on a detailed knowledge of their expression pattern, their pharmacology and the modes of signal transduction. In this context, compounds acting as agonists, antagonists or positive or negative modulators on TAARs, as well as transgenic animal models such as targeted “knock-out” mouse lines are essential tools for dissecting the molecular function of this receptor family and to fully understand their potential relevance as targets in drug development.
The present invention provides vector constructs and methods for producing non-human knock-out animals comprising within their genome a targeted deletion of the TAAR1 gene. Said TAAR1 knock-out animals, as well as methods of producing them, are also provided. The invention also relates to the use of these animals as a tool for assessing TAAR1 function and for identifying unknown ligands of TAAR 1 as well as for the characterization of novel ligands of TAARs other than TAAR1, for analyzing the tissue distribution of TAAR1, for analyzing TAAR1 signal transduction mechanisms, for analyzing the physiological function of TAAR1 in vivo, and for identifying and testing for the therapeutic effect of a compound in treating and preventing disorders comprising depression, anxiety disorders, bipolar disorder, attention deficit hyperactivity disorder, stress-related disorders, psychotic disorders such as schizophrenia, neurological diseases such as Parkinson's Disease, neurodegenerative disorders such as Alzheimer's disease, epilepsy, migraine, hypertension, substance abuse and metabolic disorders such as eating disorders, diabetes, diabetic complications, obesity, dyslipidemia, disorders of energy consumption and assimilation, disorders and malfunction of body temperature homeostasis, disorders of sleep and circadian rhythm, and cardiovascular disorders.
The resulting targeting vector pSKDT-Tar1-NLS-lacZ-PGK-Neo (bottom) comprises a genetic construct consisting of a genomic 5′ arm (5′arm), the NLS-lacZ reporter-gene, a PGK-Neo resistance-gene and a 3′ genomic arm (3′ arm). The Diphtheria toxin gene (Dipht.) is placed at the 3′ side of the construct.
A: Under the conditions of PCR protocol 5.1.1 (Table 1), a 2.72 kb PCR product (fragment 1,
B: Under the conditions of PCR protocol 5.1.2 (Table 1), a 7.33 kb PCR product (fragment 2,
C: Under the conditions of PCR protocol 5.1.3 (Table 1), a 2.86 kb PCR product (fragment 4,
M: 1 kb ladder, lane 1: Ø template (=without template), lane 2: TAAR1+/+, lane 3: TAAR1NLSlacZ/NLSlacZ.
The 1 kb DNA ladder (Invitrogen) was used as molecular weight standard.
A: PCR reactions specific for GAPDH (see above) on TAAR1+/+ and TAAR1NLSlacZ/NLSlacZ mouse brain cDNA preparations. From both cDNAs a 452 bp PCR product was amplified.
M: 50 bp ladder, lane 1: Ø template (=without template), lane 2: TAAR1NLSlacZ/NLSlacZ, lane 3: TAAR1+/+.
B: PCR reactions specific for NLSlacZ (see above) on TAAR1+/+ and TAAR1NLSlac/NLSlacZ mouse brain cDNA preparations. A 631 bp PCR product was amplified from TAAR1NLSlacZ/NLSlacZ, but not from TAAR1+/+ mouse brain cDNA.
M: 50 bp ladder, lane 1: Ø template (=without template), lane 2: TAAR1NLSlacZ/NLSlacZ, lane 3: TAAR1+/+.
C: PCR reactions specific for TAAR1 (see above) on TAAR1+/+ and TAAR1NLSlacZ/NLSlacZ mouse brain cDNA preparations. A 936 bp PCR product was amplified from TAAR1+/+, but not from TAAR1NLSlacZ/NLSlacZ mouse brain cDNA.
M: 1 kb ladder, lane 1: Ø template (=without template), lane 2: TAAR1NLSlacZ/NLSlacZ, lane 3: TAAR1+/+.
The 50 bp DNA ladder (Invitrogen; A, B) and the 1 kb DNA ladder (Invitrogen; C) were used as molecular weight standards.
The 10 bp DNA ladder (Invitrogen) was used as molecular weight standard.
Definitions
The term “Agonist” as used herein refers to a compound that binds to and forms a complex with a receptor and elicits a full pharmacological response which is specific to the nature of the receptor involved.
The term “Partial agonist” as used herein refers to a compound that binds to and forms a complex with a receptor and elicits a pharmacological response, which unlike for a full agonist, does not reach the maximal response of the receptor.
The term “Antagonist” as used herein refers to a compound that binds to and forms a complex with a receptor and acts inhibitory on the pharmacological response of the receptor to an agonist or partial agonist. Per definition the antagonist has no influence on receptor signaling in the absence of an agonist of partial agonist for that receptor.
The term “Modulator” as used herein, refers to a compound that binds to and forms a complex with a receptor, and that alters the pharmacological response of the receptor evoked by agonists or partial agonists in a quantitative manner.
“Oligonucleotide” and “nucleic acid” refer to single or double-stranded molecules which may be DNA, comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitutes for T), C, and G. The oligonucleotide may represent a coding strand or its complement. Oligonucleotide molecules may be identical in sequence to the sequence, which is naturally occurring or may include alternative codons, which encode the same amino acid as that which is found in the naturally occurring sequence (see, Lewin “Genes V” Oxford University Press Chapter 7, 1994, 171-174). Furthermore, oligonucleotide molecules may include codons, which represent conservative substitutions of amino acids as described. The oligonucleotide may represent genomic DNA or cDNA.
The term “allele” as used herein refers to any alternative form of a gene that can occupy a particular chromosomal locus.
The term “promoter” of a gene as used herein refers to the regions of DNA which control the expression of the gene. The TAAR1 promoter is substantially the promoter which controls the expression of the TAAR1 gene in a wildtype animal. Optionally, the genomic homologous sequences may comprise a part of the TAAR1 promoter or the whole TAAR1 promoter. The homologous sequences may optionally also comprise other TAAR1 regulatory elements.
The term “knock-out animal” as used herein refers to non-human animals comprising a targeted null-mutation of a gene function.
All references cited herein are hereby incorporated by reference in their entirety.
The present invention therefore provides a vector construct comprising genomic sequences homologous to upstream and downstream regions flanking the single coding exon of the TAAR1 gene suitable for homologous recombination and one or more selection marker genes.
The vector construct comprises genomic DNA sequences which are homologous to the sequences flanking the TAAR1 exon upstream and downstream on the chromosome. The lengths of the homologous sequences are chosen that it allows a targeted homologous recombination with the TAAR1 allele. The homologous sequences may have a length of about 2.5 up to about 300 kb. Preferably, the homologous sequences have a length of about 3 to about 6 kb. Preferably, the homologous sequences comprise at least a part of the TAAR1 promoter.
Preferably, the vector construct comprise additionally a reporter gene. Said reporter gene is located between the homologous TAAR1 flanking sequences. Preferably, the expression of said reporter gene is, after the integration into the genome, under the control of the TAAR1 promoter and optionally of other TAAR1 regulatory elements. The reporter gene may be selected of a group comprising LacZ or derivatives thereof, alkaline phophatase, fluorescent proteins, luciferases, or other enzymes or proteins which may be specifically detected and quantified in tissue or cells of various kinds. Preferably, the reporter gene is LacZ. Preferably, the reporter gene is at its N-terminus operably linked to a nuclear localization sequence (NLS). In a preferred embodiment, the reporter gene is inserted into the TAAR1 genomic sequence such that the endogenous start codon of the TAAR1 gene is preserved.
The selection marker gene may be a positive selection marker. The positive selection marker may be selected from the group comprising a neomycin resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a blasticidin S resistance gene, a xanthine/guanine phosphoribosyl transferase gene or a zeomycin resistance gene. The positive selection marker may be framed by recognition sites for a recombinase, which allows for excision of the positive selection marker gene after selection of successful homologous recombination events. Thereby, any effect of the expression of the positive selection marker on the expression of the reporter gene may be avoided. The recognition sites for a recombinase may be selected from the group comprising frt sites for flp recombinase and loxP sites (including mutated loxP sites) for cre recombinase. Preferably, the positive selection marker is a neomycin resistance gene.
The selection marker gene may also be an negative selection marker. The negative selection marker may be selected from, but not limited to, the group consisting of a diphtheria toxin gene and an HSV-thymidine kinase gene. Preferably, the negative selection marker is a diphtheria toxin gene.
Preferably, the vector construct comprises positive selection marker and a negative selection marker. More preferably, the vector construct comprises a neomycin resistance gene and a diphtheria toxin gene.
In a preferred embodiment, the vector construct is the vector contruct TAAR KO incorporated in the plasmid pSKDT-Tar1-NLS-PGK-Neo deposited under accession number DSMZ 17504 (Deposition date: 16 Aug. 2005). Vector construct TAAR KO is depicted in
The present invention further provides a method of producing a non-human knock-out animal, whose one or both alleles of TAAR1 gene are mutated and/or truncated in a way that less or no active TAAR1 protein is expressed comprising
In a preferred embodiment, in step (c) of the described method, a non-human knock-out animal may be produced whose one or both alleles of a TAAR1 gene comprise the TAAR1NLSlacZ allele as depicted in
In a further embodiment, the above-described method additionally comprises (d) further crossbreeding the knock-out animal produced in step (c) with an animal transgenic for the recombinase recognizing the recognition sites framing the positive selection marker gene.
Knock-out animals comprising targeted mutations are achieved routinely in the art as provided for example by the method by Joyner, A. L. (Gene Targeting. 1999, Second Edition, The Practical Approach Series, Oxford University Press, New York) and Hogan, B., et al. (Manipulating the mouse embryo. 1994, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor.).
For example, the heterozygous and/or homozygous knock-out animal of the above-described methods may be generated by selecting embryonic stem (ES) cell clones carrying the targeted TAAR1 allele as described above, verifying the targeted mutation in the recombinant embryonic stem cell clones, injecting the verified recombinant embryonic stem cells into blastocysts of wild type animals, transferring these injected blastocysts into pseudo-pregnant foster mothers, breeding chimeras resulting from the blastocysts to wild type animals, testing the offspring resulting from these breedings for the presence of the targeted mutation, breeding heterozygous animals, optionally to generate homozygous knock-out animals.
Embryonic stem cells used in the art which may also be used in the methods of this invention comprise for example embryonic stem cells derived from mouse strains such as C57BL/6, BALB/c, DBA/2, CBA/ and SV129. Preferably, embryonic stem cells derived from C57BL/6 mice are used (Seong, E et al (2004) Trends Genet. 20, 59-62; Wolfer, D. P. et al., Trends Neurosci. 25 (2002): 336-340).
The present invention further provides the non-human knock-out animal produced by any of the above described methods.
In another embodiment of the invention, a non-human knock-out animal is provided, whose one or both alleles of TAAR1 gene is mutated or truncated in a way that less or no active TAAR1 protein is expressed. Preferably, one or both alleles of the TAAR1 gene of the non-human knock-out animal are replaced with a reporter gene Preferably, the reporter gene is LacZ.
In a preferred embodiment, a non-human knock-out animal is provided whose one or both alleles of a TAAR1 gene comprise the TAAR1NLSlacZ allele as depicted in
The non-human knock-out animal may be any animal known in the art, which may be used for the methods of the invention. Preferably, the animal of the invention is a mammal, more preferred the knock-out animal of the invention is a rodent. The most preferred non-human knock-out animal is a mouse. Even more preferably, the non-human knock-out animal is a co-isogenic mutant mouse strain of C57BL/6.
The present invention also relates to descendants (=progeny) of the non-human knock-out animals as provided by the invention, obtained by breeding with the same or with another genotype. Descendants may also be obtained by breeding with the same genetic background.
The knock-out animals can be used for preparing primary cell cultures, and for the preparation of secondary cell lines derived from primary cell preparations of these animals. Furthermore, the knock-out animals can be used for the preparation of tissue or organ explants, and cultures thereof. In addition, the knock-out animal may be used for the preparation of tissue or cell extracts such as membrane or synaptosomal preparations.
The present invention further provides primary cell cultures, as well as secondary cell lines derived from the non-human knock-out animals as provided by the invention or its descendants. In addition, the present invention provides tissue or organ explants and cultures thereof, as well as tissue or cell extracts derived from non-human knock-out animals as provided by the invention or its descendants. Tissue or cell extracts are for example membrane or synaptosomal preparations.
Integration of the genetic construct into the genome can be detected by various methods comprising genomic Southern blot and PCR analysis using DNA isolated e.g. from tail biopsies of the animals.
It will be apparent to the person skilled in the art that there are a large number of analytical procedures which may be used to detect the expression of the reporter gene comprising methods at the RNA level such as for example mRNA quantification by reverse transcriptase polymerase chain reaction (RT-PCR) or by Northern blot, in situ hybridization, as well as methods at the protein level comprising histochemistry, immunoblot analysis and in vitro binding studies. Quantification of the expression levels of the targeted gene can moreover be determined by the ELISA technology, which is common to those knowledgeable in the art.
Quantitative measurement can be accomplished using many standard assays. For example, transcript levels can be measured using RT-PCR and hybridization methods including RNase protection, Northern blot analysis, and RNA dot blot analysis. Protein levels can be assayed by ELISA, Western blot analysis, and by comparison of immunohistochemically or histochemically stained tissue sections. Immunohistochemical staining, enzymatic histochemical stainings as well as immuno-electron microscopy can also be used to assess the presence or absence of the TAAR1 protein. The TAAR1 expression may also be quantified making use of the NLSlacZ reporter in the TAAR1 non-human knock-out animal using immunohistochemical or histochemical lacZ stainings on tissue sections or quantitative enzymatic lacZ assays performed with tissue homogenates or tissue extracts. Specific examples of such assays are provided below.
The knock-out animals of the invention may be further characterized by methods known in the art, comprising immunohistochemistry, electron microscopy, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) and by behavioral and physiological studies addressing neurological, sensory, and cognitive functions as well as physiologcal (e.g. metabolic) parameters. Examples of behavioral tests and physiological examinations are: Spontaneous behavior, behavior related to cognitive functions, pharmacologically-disrupted behavior, grip strength test, horizontal wire test, forced swim test, rotarod test, locomotor activity test, Prepulse inhibition test, Morris water maze test, Y-maze test, light-dark preference test, passive and active avoidance tests, marble burying test, plus maze test, learned helplessness test, stress-induced hyperthermia, measuring food consumption and development of body weight over time, measuring body temperature and energy consumption under resting and basal conditions and during heat and cold exposure, determining the thermoneutral zone, determining the food assimilation coefficient (e.g. by bomb calorimetry), determining the energy assimilation and the energy content of feces, determining the respiratory coefficient e.g. for analysis of the carbohydrate and lipid metabolism, determining the substrate utilization and energy expenditure during food restriction, determining the oxygen consumption, CO2— and heat production e.g. by indirect calorimetry, measuring the heart rate and blood pressure under resting, basal and stress conditions (e.g. by telemetry), determining the body composition (e.g. regarding water content, fat amount and fat-free mass).
A further objective of the present invention is the use of the non-human knock-out animal as described, or a primary cell culture or secondary cell lines, tissue or organ explants and cultures thereof, or tissue or cell extracts derived from said animals, as a model for identifying and testing for a therapeutic effect of a compound in disorders comprising depression, anxiety disorders, bipolar disorder, attention deficit hyperactivity disorder, stress-related disorders, psychotic disorders such as schizophrenia, neurological diseases such as Parkinson's Disease, neurodegenerative disorders such as Alzheimer's Disease, epilepsy, migraine, hypertension, substance abuse and metabolic disorders such as eating disorders, diabetes, diabetic complications, obesity, dyslipidemia, disorders of energy consumption and assimilation, disorders and malfunction of body temperature homeostasis, disorders of sleep and circadian rhythm, and cardiovascular disorders.
Additionally, these non-human knock-out animals as described above, these cell cultures, cell lines, tissue or organ explants, cultures, or tissue or cell extracts derived from said animals, may be used as a model for studying the TAAR signaling pathway.
Furthermore, these non-human knock-out animals as described above, these cell cultures, cell lines, tissue or organ explant cultures, or tissue or cell extracts derived from said animals, may be used as a tool for assessing TAAR1 function, in particular for assessing the TAAR1 function in disorders such as depression, anxiety disorders, bipolar disorder, attention deficit hyperactivity disorder, stress-related disorders, psychotic disorders such as schizophrenia, neurological diseases such as Parkinson's Disease, neurodegenerative disorders such as Alzheimer's Disease, epilepsy, migraine, hypertension, substance abuse and metabolic disorders such as eating disorders, diabetes, diabetic complications, obesity, dyslipidemia, disorders of energy consumption and assimilation, disorders and malfunction of body temperature homeostasis, disorders of sleep and circadian rhythm, and cardiovascular disorders and disorders involving catecholamine neurotransmitters.
Furthermore, these non-human knock-out animals as described above, these cell cultures, cell lines, tissue or organ explant cultures, or tissue or cell extracts derived from said animals, may also be used as a tool for determining the specificity of compounds acting on TAAR1.
In addition, these non-human knock-out animals as described above, these cell cultures, cell lines, tissue or organ explant cultures, or tissue or cell extracts derived from said animals, may be used as a tool for the identification of so far unknown ligands of TAAR1, and for the characterization of novel ligands acting on TAARs other than TAAR1.
The present invention further provides a method of testing TAAR1 agonists, TAAR1 partial agonists, TAAR1 positive or negative modulators (e.g. TAAR1 enhancer) or TAAR1 inhibitor compounds for effects other than TAAR1-specific effects which method comprises administering a TAAR1 agonist, a TAAR1 partial agonist, a TAAR1 positive or negative modulator (e.g. TAAR1 enhancer) or a TAAR1 inhibitor compound to a non-human knock-out animal as described above, or primary cell culture, or a secondary cell line, or a tissue or organ explant or a culture thereof, or tissue or organ extracts derived from said non-human knock-out animals or its descendants, and determining the effect of the compound comprising assessing neurological, sensory, and cognitive functions as well as physiological (e.g. metabolic) parameters and comparing these to the effect(s) of the same compound on wild type control animals. These neurological, sensory, and cognitive functions and physiological parameters are determined by behavior and physiological studies addressing these functions and parameters.
Control may comprise any animal, primary cell culture, a secondary cell line, a tissue or organ explant or a culture thereof, or tissue or organ extracts, wherein the TAAR1 gene is not mutated in a way, that less or no active TAAR1 protein is expressed, or wherein the animal, primary cell culture, or a secondary cell line, or a tissue or organ explant or a culture thereof, or tissue or organ extracts comprises the native TAAR1 gene. Preferably, the control is a wildtype animal.
Furthermore, the use of the non-human knock-out animal as described, or a primary cell culture, or a secondary cell line, or a tissue or organ explant or a culture thereof, or tissue or organ extracts derived from said non-human animal or it descendants is provided for testing of TAAR1 agonists, TAAR1 partial agonists, TAAR1 positive and negative modulators (e.g. TAAR1 enhancer) or TAAR1 inhibitor compounds for effects other than TAAR1-specific effects.
Effects other than TAAR1-specific effects may be any side-effects of TAAR1 agonists, TAAR1 partial agonists, TAAR1 positive and negative modulators (e.g. TAAR1 enhancer) or TAAR1 inhibitor compounds produced by its interaction with any other molecule.
The present invention further relates to a test system for testing TAAR1 agonists, TAAR1 partial agonists, TAAR1 positive and negative modulators (e.g. TAAR1 enhancer) or TAAR1 inhibitor compounds for effects other than TAAR1-specific effects comprising a non-human knock-out animal whose one or both alleles of a TAAR1 gene are mutated and/or truncated in a way that less or no active TAAR1 protein is expressed, or a primary cell culture, or a secondary cell line, or a tissue or organ explant or a culture thereof, or tissue or organ extracts derived from said non-human animal or it descendants, and a means for determining whether TAAR1 agonists, TAAR1 partial agonists, TAAR1 positive and negative modulators (e.g. TAAR1 enhancer) or TAAR1 inhibitor compounds exhibit effects other than TAAR1-specific effects.
In addition, the present invention provides a use of the non-human knock-out animal, whose one or both alleles of a TAAR1 gene are mutated and/or truncated in a way that less or no TAAR1 protein is expressed, or a primary cell culture, or a secondary cell line, or a tissue or organ explant or a culture thereof, or tissue or organ extracts derived from said non-human animal or its descendants for studying the intracellular trafficking of TAARs or of other cellular components linked to TAARs.
Furthermore, the present invention provides a use of the non-human knock-out animal, whose one or both TAAR1 alleles are replaced by a reporter gene for determining the TAAR1 expression profile. The expression profile can be readily analyzed because the reporter gene is expressed with the same spatiotemporal profile in the TAAR1 knock-out as is TAAR1 in wild type animals.
The invention further provides the knock-out animals, methods, compositions, kits, and uses substantially as described herein before especially with reference to the foregoing examples.
Having now generally described this invention, the same will become better understood by reference to the specific examples, which are included herein for purpose of illustration only and are not intended to be limiting unless otherwise specified, in connection with the figures described herein.
Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated.
Strategy for Gene Replacement of the TAAR1 Coding Sequence by a Synthetic NLS-lacZ Coding Sequence
To target TAAR1 in mouse embryonic stem cells (ES-cells) a gene-targeting vector was constructed and used to generate C57BL/6 mice with a deficiency of TAAR1. The gene-targeting vector completely replaces the TAAR1 coding region as well as about 1,5 kb genomic sequence downstream of the coding sequence with a synthetic NLS-lacZ-PGK-Neo cassette.
It is a replacement type gene targeting vector allowing for positive selection of homogenously recombined ES-cells using the Neomycin-phosphotransferase-gene (Neo) expressed under the control of the phosphoglycerate kinase promoter (PGK) (Galceran J, Miyashita-Lin E. M., Devaney E, Rubenstein J. L. R., Grosschedl R., Development 127 (2000): 469-482). To permit negative selection against ES clones carrying the targeting vector randomly integrated into the genome a diphtheria-toxin gene has been inserted into the vector outside of the TAAR1 genomic sequence (as described in Gabernet L., Pauly-Evers M., Schwerdel C., Lentz M., Bluethmann H., Vogt K., Alberati D., Mohler H., Boison D. Neurosci Lett. 373 (2005): 79-84).
At the same time the lacZ reporter-gene (Galceran J, Miyashita-Lin E. M., Devaney E, Rubenstein J. L. R., Grosschedl R., Development 127 (2000): 469-482) was fused to a nuclear signal sequence (NLS) and placed under the transcriptional control of the putative TAAR1 promoter and regulatory elements. Hereby the start-codon of the synthetic reporter is identical to the start-codon of TAAR1. This allows the sensitive analysis of the expression pattern conferred by the endogenous TAAR1 control region in histochemical stainings for the product of the lacZ gene.
Cloning of a Plasmid for Targeting of the NLS-lacZ Coding Sequence to the TAAR1 Gene by Homologous Recombination in ES-cells
Construction of the TAAR1 gene Targeting Vector
The resulting targeting vector consists of a genomic 5′ arm (5′ arm), the NLS-lacZ reporter-gene, a PGK-Neo resistance cassette and a 3′ genomic arm. The Diphtheria toxin cassette (Dipht.) is placed at the 3′ side of the targeting vector (see
Oligonucleotides were designed based on the published genomic sequences of the mouse TAAR1 locus (Mouse genome sequence database, NCBI draft 34, May 2005) and obtained from a commercial supplier (Microsynth AG, Balgach, Switzerland). All molecular cloning techniques were carried out essentially according to Sambrook et. al. (Molecular Cloning: A laboratory manual. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.) and the instructions of the suppliers of kits and enzymes.
The targeting vector pSKDT-Tar1-NLS-lacZ-PGK-Neo contains 4.0 kb genomic sequence 5′ of the mouse TAAR1 coding sequence and 1.7 kb genomic sequence 3′ of the mouse TAAR1 coding sequence (
These sequences were amplified from genomic C57BL/6 DNA using proofreading PCR and cloned into cloning vectors.
To clone the 5′ arm oligonucleotide mTar1-5′-KpnI-16c and oligonucleotide mTar1-755-nc were used in the following PCR reaction.
2 ng/μl genomics C57BL/6 DNA, 200 μM dNTPs (PCR Nucleotide Mix, Roche Diagnostics, Mannheim, Germany), 0,5 μM of each oligonucleotide, 1×PCR buffer (Promega, Madison, Wis., USA), 0,06 U/μl Pfu Polymerase (Promega, Madison, Wis., USA) in a total volume of 100 μl were incubated with the following protocol: 95° C. 2 min., 35× (95° C. 45 sec., 59° C. 45 sec., 72° C. 9 min.), 72° C. 7 min, ∞ 4° C. on a PCR Thermocycler MJ Research PTC-200 (MJ Research Inc., Watertown, USA). The resulting PCR product of 4,702 kb contained the 5′ arm of the TAAR1 locus and was cloned into the Srfl site of pPCR-Script Amp SK+ (Invitrogen-Gibco, Carlsbad, Calif., USA).
Orientation and parts of the sequence were confirmed by sequencing using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and an ABIPrism 310 Genetic Analyzer.
The resulting vector is pPCR-Script-5′Tar1.
To clone the 3′ arm oligonucleotide mTar1-33c and oligonucleotide mTar1-SmaI-11nc were used in the following PCR reaction.
2 ng/μl genomics C57BL/6 DNA, 200 μM dNTPs (PCR Nucleotide Mix, Roche Diagnostics, Mannheim, Germany), 0,5 μM of each oligonucleotide, 1×PCR buffer (Promega, Madison, Wis., USA), 0,06 Uμl Pfu Polymerase (Promega, Madison, Wis., USA) in a total volume of 100 μl were incubated with the following protocol: 95° C. 2 min., 35× (95° C. 45 sec., 60° C. 45 sec., 72° C. 4 min.), 72° C. 7 min, ∞ 4° C. on a PCR Thermocycler MJ Research PTC-200 (MJ Research Inc., Watertown, USA).
The resulting PCR product of 1651 kb was cloned into the Srfl site of pPCR-Script Amp SK+ (Invitrogen-Gibco, Carlsbad, Calif., USA).
Orientation and parts of the sequence were confirmed by sequencing using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and an ABIPrism 310 Genetic Analyzer.
The resulting vector is pPCR-Script-3′Tar1.
The targeting vector was assembled in 3 steps.
Subsequently, the 5′ genomic arm cloned as described above was removed from the plasmid pPCR-Script-5′Tar1 by restriction digest with NsiI and KpnI and subsequent agarose gel electrophoresis and gel extraction. Following, the 4 kb genomic DNA fragment was ligated into the plasmid pSKDT-3′Tar1, which previously had been digested with NsiI and KpnI, resulting in the plasmid pSKDT-5′-3′Tar1
Step 2: A synthetic sequence harboring several restriction sites (see
Step 3: The NLS-lacZ-PGK-Neo cassette was inserted into the plasmid pSKDT-5′-3′Tar1-NLS. For this purpose plasmid pSKDT-5′-3′Tar1-NLS was linearized with a Stul restriction digest. The NLS-lacZ-PGK-Neo cassette was isolated from the plasmid C8βgal (Galceran J, Miyashita-Lin E. M., Devaney E, Rubenstein J. L. R., Grosschedl R. Hippocampus development and generation of dendate gyrus granule cells is regulated by LEF1. Development 127 (2000): 469-482) with a Smal restriction digest and subsequent agarose gel electrophoresis and gel extraction. The NLS-lacZ-PGK-Neo cassette DNA fragment was ligated into the StuI linearized plasmid pSKDT-5′-3′Tar1-NLS, resulting in the targeting vector pSKDT-Tar1-NLS-lacZ-PGK-Neo.
Deposition data: The plasmid pSKDT-Tar1-NLS-lacZ-PGK-Neo comprising the genetic construct TAAR1-KO (see
Gene Targeting of the TAAR1 Gene in Mouse ES Cells by Homologous Recombination
Culturing of C57BL/6 ES-cells
Handling of ES-cells was performed essentially as described in Joyner (Gene Targeting. 1999, Second Edition, The Practical Approach Series, Oxford University Press, New York). C57BL/6 ES-cells (Eurogentec, Seraing, Belgium) were grown on monolayers of mitotically inactivated primary mouse embryonic fibroblast (MEF) cells isolated from a mouse line CD1-Tg.neoR expressing the neomycin resistance gene (Stewart C. L., Schuetze S., Vanek M., Wagner E. F. Expression of retroviral vectors in transgenic mice obtained by embryo infection. EMBO J. 6 (1987): 383-8). MEFs were isolated as described in (Joyner, A L, eds.: Gene Targeting. A Practical Approach. 2000, Oxford University Press, New York) and mitotically inactivated by gamma radiation (18Sv in a Cs-137 irradiation source).
ES-cells were grown in ES-medium containing Dulbeccos's modified Eagle Medium (Invitrogen-Gibco, Carlsbad, Calif., USA) supplemented with 15% FCS (Inotech/Biological Industries, Beit Haemek, Israel), 100 IU/ml Penecillin/Streptomycin (Invitrogen-Gibco, Carlsbad, Calif., USA), 0.5 mM β-Mercaptoethanol (Invitrogen-Gibco, Carlsbad, Calif., USA), non essential amino acids MEM (1x, Invitrogen-Gibco, Carlsbad, Calif., USA), 2 mM Glutamine (Invitrogen-Gibco, Carlsbad, Calif., USA) and 1000 U/ml leukocyte inhibitory factor (Chemicon, Temecula, Calif., USA).
Electroporation of the Targeting Vector into ES-cells
The SacII linearized targeting vector pSKDT-Tar1-NLS-lacZ-PGK-Neo (total amount: 30 μg) was added to 30×106 ES-cells in a buffer containing 137 mM NaCl, 2.7 mM KCl, 90 mM Na2HPO4, 1,5 mM KH2PO4, pH 7.4 (PBS) and electroporated with a Bio-Rad Genepulzer with a capacity extender (Bio-Rad, Hercules, Calif., USA; settings: 280 V, 500 μF). Thereafter, ES-cells were plated on MEF mono cell layers and selected for the presence of the Neomycin gene in ES-medium supplemented with 350 μg/ml G418 (geneticin, Sigma-Aldrich, St. Louis, Mo., USA).
Individual, well separated ES-clones originating from the transfected cells were transferred into 48 well culture dishes and grown for 10 days. ⅓ of the cells were used to isolate genomic DNA using the MagNAPure LC system (Roche Diagnostics, Basel, Switzerland; Laird, P. W., Zijderveld, A., Linders, K., Rudnicki, M. A., Jaenisch, R., Berns, A.: Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 1991; 19 (15): 4293) and analyzed using PCR. The remaining ⅔ of the cells were eventually used for further analysis and for blastocyst injections.
2.2.3) Screening of Monoclonal ES-cells with PCR for Correct Targeting of TAAR1
In 62 clones the correct gene targeting event was assessed by PCR amplification of DNA fragments spanning the transition points between the genomic DNA included into the targeting vector and surrounding genomic sequence.
To test for correct recombination at the 3′arm PCR clones were screened with PCRs using oligonucleotides IL4-neo and mTar26nc in the following protocol:
This PCR yields PCR products only in the presence of genomic DNA of ES-clones, in which the correct homologous recombination event between the 3′ arm of the targeting vector and the chromosomal DNA as depicted in
Importantly clone IIB1 gives amplification at the expected size of 3.2kb (
To test for correct recombination at the 5′ arm PCR clones were screened with PCRs using oligonucleotides IL4-rev and mTar24c in the following protocol:
This PCR yields PCR products only in the presence of genomic DNA of ES clones, in which the correct homologous recombination event between the 5′ arm of the targeting vector and chromosomal DNA as depicted in
The above PCR conducted with genomic DNA of Clone IIB1 gives an amplification at the expected size of 9.8kb (
Clone IIB1 was chosen for injection into blastocysts as described in chapter 3.
Following Oligonucleotides (all sequences in 5′→3′ orientation) were used:
Generation of a TAAR1-NLSlacZ Knock-in Mouse Line from a Mutant ES cell Line
All handling of animals was carried out in compliance with Swiss Federal and Cantonal laws on animal research, and permission for the generation and handling of the mutant mouse line was specifically granted from the Kantonale Veterinaramt of Basel City with the Tierversuchgenehmigung No. 2055 as of Aug. 23rd 2004.
A mutant mouse line which carries an inheritable targeted mutation of the TAAR1 gene as described in chapter 1) in all cells was generated following standard procedures essentially as described in Hogan et al. (Manipulating the mouse embryo. 1994, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor.) by Polygene AG (Rümlang, Switzerland). For the generation of chimeric animals, the monoclonal mutant ES cells (clone IIB1, as described in chapter 2) were injected into embryonic day 3.5-4.5 (E3.5-4.5) Balb/c blastocysts and transferred into pseudo-pregnant C57BL/6×CBA F1 females. Offspring delivered by these females were judged for the degree of chimerism (i.e. the degree of overall contribution of ES cells to the chimeric animals). The employed breeding paradigm allowed to use the coat color as parameter for estimating the degree of chimerism, with the percentage of black hairs in the fur reflecting the degree of chimerism.
Male high percentage chimeras were naturally mated with C57BL/6 females, and all offspring with purely black coat color was analyzed for the presence of the TAAR1NLSlacZ allele. To this end, genomic DNA was isolated from tail biopsies of adult animals with a MagNAPure LC system for nucleic acid purification (Roche Applied Science, Rotkreuz, Switzerland) according to the instructions of the manufacturer and analyzed for the presence of the TAAR 1 wild type allele (TAAR1+; indicating the presence of the undisrupted TAAR1 gene) as well as the neomycin phosphotransferase coding sequence (indicating the presence of the TAAR1NLSlacZ allele) by means of PCR.
PCR reactions were performed on a Gen Amp 9700 thermocycler (Applied Biosystems, Rotkreuz, Switzerland) in a total volume of 50 μl per reaction composed as follows (final concentrations/amounts):
TAAR1+ allele:
TAAR1NLSlacZ allele:
The PCR reactions were run with the following temperature profile: 95° C. 2min., 35× (95° C. 30 sec., 57° C. 30 sec., 72° C. 1 min.), 72° C. 5 min, ∞4° C. the PCR products were analyzed by standard agarose gel electrophoresis as described in Sambrook et al. (Molecular Cloning: A laboratory manual. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.), and the expected DNA fragment sizes were 755 bp for the undisrupted TAAR1 allele and 280 bp for the TAAR1NLSlacZ allele.
Characterization of the TAAR1-NLSlacZ Knock-out
Analysis of the TAAR1-NLSlacZ gene Replacement Inherited in the TAAR1-NLSlacZ Knock-out Mouse Line
In order to proof that the TAAR1NLSlacZ allele equals the designed mutant gene as described in 2 the targeted gene locus was analyzed based on genomic DNA derived from homozygous mutants of the F2 generation. The correct gene targeting event was verified by PCR amplification and DNA sequence analysis of DNA fragments spanning the transition points between the genomic DNA included into the targeting vector and surrounding genomic sequence (see
The DNA fragments 1-4 (see
20-100 ng genomic DNA, 1× concentrated PCR buffer 3 (part of Expand High Fidelity PCR System; Roche Diagnostics, Mannheim, Germany), 500 μM of each dNTP (Amersham), 300 nM of each oligonucleotide (Microsynth AG) and 3.75 U/reaction Expand High Fidelity Enzyme Mix (part of Expand High Fidelity PCR System; Roche Diagnostics). The PCR reactions were run using the combinations of genomic DNA template, oligonucleotide and PCR protocols as summarized in Table 1.
The PCR reactions were run with one of the following temperature protocols:
Protocol 5.1.1:
The PCR products were analyzed by standard agarose gel electrophoresis (
For DNA sequence analysis DNA fragments 1-4 were amplified from genomic DNA by PCR and subjected to agarose gel electrophoresis as described above. PCR products of the expected sizes were cut out from the gels with sterile scalpels (Bayha, Tuttlingen, Germany) and extracted from the agarose gel slices using the QIAquick Gel Extraction Kit (QIAGEN AG, Basel, Switzerland) following the instructions of the manufacturer. The extracted PCR products were adjusted to a defined concentration with cold ethanol/sodium acetate DNA precipitation (Sambrook, J., Fritsch, E. F., und Maniatis, T.: Molecular Cloning: A laboratory manual. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.) and subsequently dissolving the DNA pellets in the required volume of 10 mM Tris/HCl pH 7.5. DNA sequence analysis of the PCR products was carried out at Microsynth AG employing BigDye chemistry and a 3730 capillary DNA analyzer (Applied Biosystems), and the oligonucleotides used for the individual sequence analysis reactions as well the sequence analysis results are summarized in listing 1-9 (see below). The sequence listings are limited to those parts of the sequence analysis results which were judged as reliable based on the chromatograms.
Listing 1 (SEQ. ID NO: 1): Result of DNA sequence analysis of DNA fragment 1 (
Listing 2 (SEQ. ID NO: 2): Result of DNA sequence analysis of DNA fragment 2 (
Listing 3 (SEQ. ID NO:3): Result of DNA sequence analysis of DNA fragment 2 (
Listing 4: (SEQ. ID NO: 4) Result of DNA sequence analysis of DNA fragment 2 (
Listing 5 (SEQ. ID NO: 5): Result of DNA sequence analysis of DNA fragment 3 (
Listing 6 (SEQ. ID NO: 6): Result of DNA sequence analysis of DNA fragment 3 (
listing 7 (SEQ. ID NO: 7): Result of DNA sequence analysis of DNA fragment 4 (
Listing 8 (SEQ. ID NO: 8): Result of DNA sequence analysis of DNA fragment 4 (
Listing 9 (SEQ. ID NO: 9): Result of DNA sequence analysis of DNA fragment 4 (
In order to confirm the successful gene replacement on the transcript level cDNA derived from whole brains of juvenile TAAR1+/+ or TAAR1NLSlacZ/NLSlacZ mice were prepared as described below.
RNA was prepared from tissue samples essentially according to Chomczynski and Sacchi (Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156-159.). In brief, mouse pups of day 11 after birth were sacrificed, and total brains were removed and homogenized in the 10× volume of Trizol Reagent (Invitrogen, Paisley, UK) in a glass douncer (Inotech AG, Dottikon, Switzerland). Total RNA was isolated according to the instructions of the manufacturer. Raw RNA was dissolved in 10 mM Tris/HCl pH 7.0 and treated with 25 U/μl DNAse I (Roche Diagnostics, Rotkreuz, Switzerland) in 10 mM Tris/HCl pH 7.0 with 10 mM MgCl2 for 1 hr. The DNAse I was removed by phenol/chloroform extraction according to (Sambrook, J., Fritsch, E. F., und Maniatis, T.: Molecular Cloning: A laboratory manual. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). RNA was precipitated, dissolved to a final concentration of 1 mg/ml in 10 mM Tris/HCl pH 7.0 and used for first strand synthesis.
For cDNA synthesis Superscript II RNAse H- Reverse Transcriptase (Invitrogen, Paisley, UK) was used according to the instructions of the manufacturer. Briefly, 4 μg total RNA were mixed with 1 μg oligo (dT)12-18 (Invitrogen, Paisley, UK) in H2O in a total volume of 23 μl, incubated at 70° C. for 10 min. and chilled on ice. The following components were added on ice (final concentrations): 50 mM Tris-HCl (pH8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM of each dNTP (Amersham, Otelfingen, Switzerland), 1 U/μl RNAse Out RNAse inhibitor (Invitrogen) and 10 U/μl reverse transcriptase. The reaction was incubated for 1 hr at 42° C., stopped by incubation at 70° C. for 10 min. and diluted to a total volume of 100 μl with 10 mM Tris/HCl pH 7.0.
PCR amplification of transcripts was carried out essentially as described in 4, but with the following modifications: PCR reactions were carried out in a total volume of 50 μl per reaction composed as follows (final concentrations/amounts): 1 μl of cDNA preparation (equivalent to a total amount of 40 ng whole brain total RNA), 20 mM Tris-HCl (pH8.4), 50 mM KCl, 1.5 mM MgCl2, 200 nM of each oligonucleotide (Microsynth AG, Balgach, Switzerland), 200 mM of each dNTP (Amersham), 5 U/reaction recombinant Taq DNA polymerase (Invitrogen). For the detection of individual mRNA transcripts the following oligonucleotides and temperature profiles were used:
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH):
Oligonucleotides:
Temperature Profile:
Oligonucleotides:
Oligonucleotides:
As expected, the GAPDH transcript was detected in both cDNA preparations from TAAR130 /+ and TAAR1NLSlacZ/NLSlacZ mouse brain indicating that the cDNA preparations per se were successful. While the PCR analysis detected TAAR1 transcript only in TAAR1+/+, but not in TAAR1NLSlacZ/NLSlacZ mouse brain cDNA, the NLSlacZ transcript was detected only in TAAR1NLSlacZ/NLSlacZ, but not in TAAR1+/+ mouse brain cDNA.
These data reveal that there is no TAAR1 expression in TAAR1NLSlacZ/NLSlacZ mouse brain which is in agreement with the deletion of the TAAR1 coding sequence in the TAAR1NLSacZ/NLSlacZ mouse line. The presence of NLSlacZ mRNA transcript in TAAR1NLSlacZ/NLSlacZ mouse brain provides evidence for the expression of NLSlacZ in TAAR1NLSlacZ/NLSlacZ mutants from the endogenous TAAR1 gene locus. The absence of NLSlacZ transcript from TAAR1+/+ mouse brain as apparent from
1.1) Analysis of the Genetic Background of the TAAR1-NLSlacZ Knock-in Mouse Line
The genetic background of genetically modified mouse lines has a profound impact on their phenotype, and the variability of the genetic background between individual animals of a mouse line caused e.g. by a so-called mixed genetic background can complicate or even make impossible the meaningful and consistent phenotypical characterization of a mutant mouse line or its use e.g. in behavioral pharmacology (Gerlai, R., Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci. 19 (1996) 177-181.; Bucan and Abel, The mouse: genetics meets behaviour. Nat. Rev. Genet. 3 (2002)114-123; Banburry Conference on Genetic Background in Mice (1997): Mutant Mice and Neuroscience: Recommendations Concerning Genetic Background. Neuron 19, 755-759). For historical and practical reasons, targeted mouse mutants are most frequently generated using ES cells derived from one of the various SV129 inbred mouse lines (Hogan, B., Beddington, R., Costantini, F., und Lacy, E.: Manipulating the mouse embryo. 1994, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor.; Threadgill, D. W., Yee, D., Matin, A., Nadeau, J. H., Magnuson ,T., Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain. Mamm Genome. 8 (1997) 390-393). Because of the unfavorable properties of the SV129 mouse lines related to neuroanatomy, breeding performance and behavior mice generated using SV129 ES cells need to be transferred to a homogenous and more favorable genetic background by backcrossing with mice of the desired genetic background for at least 10 generations requiring several years of work (Silver, L. M.: Mouse Genetics. 1995, Oxford University Press, New York).
The use of ES cells derived from C57BL/6 mice (Kontgen, F., Suss, G., Stewart, C., Steinmetz, M., Bluethmann H., Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int Immunol. 5 (1993) 957-964) in combination with an appropriate breeding scheme allowed us to generate a mutant mouse line carrying the TAAR1NLSlacz allele on a pure C57BL/6 genetic background. The homogeneity of the genetic background was experimentally confirmed by means of microsatellite analysis on 5 heterozygous mutants of the F1 generation as well as for the ES cell line used for the generation of germline chimeras.
For this analysis genomic DNA was isolated from tail biopsies of 5 mice of the F1 generation carrying the TAAR1NLSlacZ allele, as well as from a sample of ES cells used for the generation of the germline chimeras, with the MagNAPure LC system for nucleic acid purification (Roche Diagnostics, Basel, Switzerland). Genomic DNA of congenic C57BL/6, DBA and SV129 mice were used as standard and were analyzed in parallel by PCR; the genomic DNA of the congenic inbred mouse strains C57BL/6, DBA and SV129 were purchased from Jackson Laboratory (Bar harbor, Me. USA).
PCR reactions were performed on a GenAmp 9700 thermocycler Applied Biosystems) in a total volume of 20 μl per reaction composed as follows (final concentrations/amounts):
For confirming the homogeneity of the genetic background of the mutant mouse line carrying the TAAR1NLSlacZ allele a density of about 2 markers pro chromosome were used, and the following microsatelites were included into the analysis: D1MIT217, D1MIT291, D2MIT312, D2MIT285, D3MIT22, D3MIT45, D4MIT149, D4MIT166, D5MIT259, D5MIT95, D6MIT86, D6MIT188, D7MIT76, D7MIT246, D8MIT155, D8MIT248, D9MIT191, D9MIT182, D1MIT35, D10MIT83, D11MIT149, D11MIT99, D12MIT136, D12MIT99, D13MIT16, D13MIT35, D14MIT203, D14MIT165, D15MIT193, D16MIT131, D16MIT4, D17MIT93, D17MIT155, D18MIT19, D18MIT152, D19MIT71, DxMIT64 (oligonucleotide sequences were chosen according to Jaxon Lab Mouse Informatics Database; Eppig, J. T. et al. The Mouse Genome Database (MGD): from genes to mice—a community resource for mouse biology. Nucleic Acids Res. 33, D471-D475 2005).
The microsatellite analysis revealed that the mutant mouse line carrying the TAAR1NLSlacZ allele matches with wild type C57BL/6 mice for all microsatellites tested (see
1.2) Proof of Concept: Use of TAAR1-NLSlacZ Gene Replacement as Tool for Analyzing the Tissue Distribution of TAAR1 Expression
The TAAR1NLSlacZ mutant mouse line was generated using a gene replacement strategy as described in chapter 1). As a consequence, the histological marker NLSlacZ has been targeted to the TAAR1 gene locus such that its expression reflects the spatio-temporal tissue distribution of TAAR1 expression in wild type animals. To this end, the TAAR1NLSLacZ mutant mouse line serves as a powerful tool which allows for detailed TAAR1 expression studies without the need to generate and validate TAAR1-specific probes such as specific antibodies or radioligands. The expression of a synthetic coding sequence from a chromosomal locus can be potentially compromised e.g. by gene silencing events or by insufficient expression levels, both of which are difficult to predict without experimental data derived from the actual mutant of interest. In order to proof the functionality of the NLSlacZ coding sequence targeted to the TAAR1 gene locus in the TAAR1NLSlacZ mutant mouse as histological marker a lacZ staining was carried out on tissue sections of adult TAAR1NLSlacZ/NLSlacZ and TAAR1+/+ mouse brains.
Adult TAAR1NLSacZ/NLSlacZ and TAAR1+/+ mice were transcardially perfused under terminal isoflurane anesthesia essentially as described in Romeis (Mikroskopische Technik. 1989, 17., neubearbeitete Auflage, Urban and Schwarzenberg; Müchen, Wien, Baltimore). The animals were perfused consecutively with 10 ml phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 90 mM Na2HPO4, 1,5 mM KH2PO4, pH 7.4) and 15 ml fixative (2% w/v paraformaldehyde and 0.2% w/v glutaraldehyde in PBS). The brains were removed from the skull, post-fixed for 4 hours in fixative at 4° C. and immersed into 0.5 M sucrose in PBS over night at 4° C. Brains were embedded in OCT compound (Medite Medizintechnik, Nunningen, Switzerland) in Peel-A-Way tissue embedding molds (Polysciences Inc., Warrington, USA) and frozen on liquid nitrogen. Brains were cut in parasagittal orientation on a cryomicrotome (Leica Microsystems AG, Glattbrugg, Switzerland) at 50 μm and thaw mounted on gelatin coated glass slides (Fisher Scientific, Wohlen, Switzerland). Tissue sections were air dried at room temperature for 2 h, washed 5 times for 10 min each in PBS at room temperature (RT) and incubated for 16-24 h in lacZ staining solution (1 mg/ml 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2 in PBS) in a light tight container at 37° C. on a horizontal shaker. The staining process was stopped by washing the tissue sections 5 times for 10 min each in PBS at RT. Tissue sections were dehydrated through an ascending ethanol series, equilibrated to xylene and coverslipped with DePex (Serva GmbH, Heidelberg, Germany). Tissue sections were analyzed on an Axioplan I microscope (Carl Zeiss AG, Feldbach, Switzerland) equipped with an Axiocam CCD camera system (Carl Zeiss AG).
The lacZ staining of the tissue sections revealed a strong and specific staining in TAAR1NSLlacZ/NLSlacZ mouse brain sections, which was absent from a tissue section of an equivalent region of a TAAR1+/+ mouse brain. This result confirms, that NLSlacZ expression from the TAAR1 gene locus in TAAR1NLSlacZ mutant mice functions as histological marker for analyzing TAAR1 expression in mouse (
In order to exclude potential deficits in the spontaneous behavior or sensory capabilities and the physiological conditions of the TAAR1NLSlacZ mutant mouse line, which could arise through the mere presence of the targeted mutation of the TAAR1 gene. The baseline conditions were analyzed in adult animals 3 months of age of all three genotypes and both genders.
To this end, the physical state of animals was examined regarding gain of body weight in the first three months of postnatal age, regarding rectal temperature, and nest building behavior. The neurological state of the animals was analyzed regarding the potential occurrence of catalepsy, ataxia, tremor, lacrimation and salivation and the degree of arousal in response to transfer of the animals to a novel environment. The dexterity and coordination of the animals was examined by analyzing grip strength and spontaneous horizontal locomotor activity as well as in the so-called rotarod and horizontal wire tests (e.g. Crawley, J. N. (2000) What's Wrong With My Mouse? 1. Edition, Wiley & Sons, ISBN 0471316393; Irwin, S. Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia 13, 222-257, 1968).
None of the tests and examinations revealed a significant difference in comparison of the genotypes, demonstrating that there are no deficits in spontaneous behavior, sensory capabilities or physiological state which could impact the characterization of the mutant mouse line in behavioral models directed towards dissecting the potential role of TAAR1 in disease models or towards the characterization of pharmacological compounds.
Behavioral Phenotyping
Animals were maintained under conditions of constant temperature (22±2° C.) and humidity (55-65%), and all mice were singly housed for the duration of study. Food and water were available ad libitum. All experiments were conducted during the light phase of the light/dark cycle (lights on: 6 a.m.-6 p.m.). All animal procedures were conducted in strict adherence to the Swiss federal regulations on animal protection and to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and with the explicit approval of the local veterinary authority.
Behavioral assessment. Mice were assessed for standard physiological parameters (body temperature, evolution of body weight), and in several neurological and behavioral tests, including grip strength (g), horizontal wire test, rotarod test, and locomotor activity.
Body temperature was measured to the nearest 0.1° C. by a HANNA instruments thermometer (Ronchi di Villafranca, Italy) by inserting a lubricated thermistor probe (2 mm diameter) 20 mm into the rectum; the mouse was hand held at the base of the tail during this determination and the thermistor probe was left in place until steady readings were obtained (˜15 s).
Body weight (g) was checked at various time points in mice aged from 12 to 24 weeks.
Horizontal wire test: mice were held by the tail and required to grip and hang from a 1.5 mm in diameter bar fixed in a horizontal position at a height of 30 cm above the surface for a maximum period of 1 min. The latency for the mice to fall was measured, and a cut-off of either 60 sec or the highest fall latency score from three attempts was used.
Rotarod test: The apparatus consisted of a fixed speed rotarod (Ugo Basile, Biological Research Apparatus, Varese, Italy) rotating at either 16 or 32 rpm. Bar is 10 cm wide, 3 cm in diameter, and 25 cm above the bench. Motor incoordination on the rotating rod translates into animals falling off the bar. On the test day, subjects were placed on the rotarod and their latency to fall measured. All mice were tested at both 16 and 32 rpm, and in both tests a cut-off of either 120 s or the highest fall latency score from three attempts was used.
Locomotor activity. A computerized Digiscan 16 Animal Activity Monitoring System (Omnitech Electronics, Colombus, Ohio) was used to quantitate spontaneous locomotor activity. Data were obtained simultaneously from eight Digiscan activity chambers placed in a soundproof room with a 12 hr light/dark cycle. All tests were performed during the light phase (6 a.m. to 6 p.m.). Each activity monitor consisted of a Plexiglas box (20×20×30.5 cm) with sawdust bedding on the floor surrounded by invisible horizontal and vertical infrared sensor beams. The cages were connected to a Digiscan Analyzer linked to a PC that constantly collected the beam status information. With this system, different behavioral parameters could be measured, such as horizontal and vertical activity, total distance travelled (in cm), and stereotypies. The mice were tested via a pseudo-Latin squares design twice weekly with at least a 10-day interval between two consecutive test sessions. Vehicle (saline 0.9%) or d-amphetamine (0. 4, 1, 2.5, 5 mg/kg, i.p.) was administered to wild-type (n=16) and TAAR-1 KO (n=12) mice just prior to testing. Locomotor activity was recorded for 90 min starting immediately after the mice were placed in the cages.
Statistics. Behavioral observations were recorded as mean values SE and analyzed with an unpaired t test. Locomotor activity data (total distance) were analyzed with a two-factor (Genotype and Dose) ANOVA with repeated measures. Comparisons of dose effects in each genotype were undertaken with a repeated measures ANOVA, followed in significant cases by paired t tests. A p value of 0.05 was accepted as statistically significant.
Assessment of Extracellular Levels of Biogenic Amines
Four months old male mice were used for these experiments. The procedures used for the experiments described in this report received prior approval from the City of Basel Cantonal Animal Protection Committee based on adherence to federal and local regulations on animal maintenance and testing.
Surgery and Implantation of the Microdialysis Probe
Forty-five minutes before anesthesia mice received subcutaneously 0.075 mg/kg of buprenorfine. Mice were then anesthetized with isoflurane and placed in a stereotaxic device equipped with dual manipulators arms and an anesthetic mask. Anesthesia was maintained with isoflurane 0.8-1.2% (v/v; support gas oxygen/air, 2:1). The head was shaved and the skin was cut along the midline to expose the skull. A small bore hole was made in the skull to allow the stereotaxical insertion of the microdialysis probe (vertical probe carrying a 2 mm polyacrilonitrile dialysis membrane; Brains On-line, Groningen, The Netherlands) in the striatum (coordinates: A 0.9 mm, L-1.8 mm, V-4.6 mm). The probe was cemented into place using binary dental cement. Once the cement was firm, the wound was closed with silk thread for suture (Silkam) the animal was removed from the stereotaxic instrument and returned to its cage. At the end of the surgery and 24 hrs later mice were treated with Meloxicam 1 mg/kg sc. The body weight of the animals was measured before the surgery and in the following days to monitor the recovery of the animal from surgery.
Microdialysis Experiments
All microdialysis experiments were carried out 3-4 days after surgery in awake, freely moving mice. The day of the experiment, the inlet of the implanted dialysis probe was connected to a micro-perfusion pump (CMA/Microdialysis, Sweden) and the outlet was connected to a fraction collector. The microdialysis probe was then perfused with Ringer solution (NaCl 147 mM, KCl 3 mM, CaCl2 1.2 mM, MgC12 1.2 mM) at a constant flow rate of 1.5 μL/min and 0.02 M Four samples of dialysates were collected before pharmacological treatment to determine the baseline levels of biogenic amines and their metabolites. Mice were then treated intraperitoneally with 2.5 mg/kg of amphetamine and dialysate samples collected for further 2.5 hrs. Dialysate samples were stored frozen at −80° C. until analysis.
Analysis of Microdialysate
Frozen dialysate samples were shipped in dry ice to Brains On-Line for assay of monoamines and their metabolites. The concentrations of dopamine, DOPAC, serotonin, 5-HIAA and noradrenaline were measured by use an HPLC equipped with an electrochemical detector according to the procedure of van der Vegt et al. (2003).
In Vivo Microdialysis Assessment of Biogenic Amine Neurotransmitter Levels
Concentrations of norepinephrine, dopamine, and serotonin were determined within the same samples by HPLC separation and electrochemical detection. Samples were split into two aliquots; one used for simultaneous analysis of norepinephrine and dopamine, the other for analysis of serotonin.
Norepinephrine and Dopamine
Aliquots (20 μL) were injected onto the HPLC column by a refrigerated microsampler system, consisting of a syringe pump (Gilson, model 402), a multi-column injector (Gilson, model 233 XL), and a temperature regulator (Gilson, model 832). Chromatographic separation was performed on a reverse-phase 150×2.1 mm (3 μm) C18 Thermo BDS Hypersil column (Keystone Scientific). The mobile phase (isocratic) consisted of a sodium acetate buffer (4.1 g/L Na acetate) with 2.5% v/v methanol, 150 mg/L Titriplex (EDTA), 150 mg/L 1-octanesulfonic acid, and 150 mg/L tetramethylammonium chloride (pH=4.1 adjusted with glacial acetic acid). Mobile phase was run through the system at a flow rate of 0.35 mL/min by an HPLC pump (Shimadzu, model LC-10AD vp).
Norepinephrine and dopamine were detected electrochemically using a potentiostate (Antec Leyden, model Intro) fitted with a glassy carbon electrode set at +500 mV vs. Ag/AgCl (Antec Leyden). Data were analyzed by Chromatography Data System (Shimadzu, class-vp) software. Concentrations of monoamines were quantitated by external standard method.
Serotonin
Aliquots (20 μL) were injected onto the HPLC column as described for norepinephrine and dopamine. Chromatographic separation was performed on a reverse-phase 100×2 mm (3 μm) C18 ODS Hypersil column (Phenomenex). The mobile phase (isocratic) consisted of a sodium acetate buffer (4.1 g/L Na acetate) with 4.5% v/v methanol, 500 mg/L Titriplex (EDTA), 50 mg/L 1-heptanesulfonic acid, and 30 μL/L tetraethylammonium (pH=4.74 adjusted with glacial acetic acid). Mobile phase was run through the system at a flow rate of 0.4 mL/min by an HPLC pump (Shimadzu, model LC-10AD vp). Serotonin was detected electrochemically using the same method as described for norepinephrine and dopamine.
Slice Electrophysiology in the Ventral Tegmental Area (VTA).
Horozontal slices (250 μm thick, VT1000 vibratome, Leica) of the midbrain were prepared from TAAR1 knock-out and littermate wild-type mice 25-60 days of age. Slices were cooled in artificial cerebrospinal fluid (ACSF) containing in mM: 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1.0 NaH2PO4, 26.2 NaHCO3 and 11 glucose. Slices were continuously bubbled with 95% O2 and 5% CO2 and transferred after 1 h to the recording chamber superfused (1.5 ml/min) with ACSF at 32-34° C. The VTA was identified as the region medial to the medial terminal nucleus of the accessory optical tract. Visualized whole-cell current-clamp recording techniques were used to measure the spontaneous firing rate and holding currents of neurons. All cells used for the statistical analysis displayed a stable firing activity for more than 30 minutes. Dopaminergic neurons were identified by a large Ih current. The internal solution contained in mM: 140 potassium gluconate, 4 NaCl, 2 MgCl2, 1.1 EGTA, 5 HEPES, 2 Na2ATP, 5 sodium creatine phosphate and 0.6 Na2GTP; the pH was adjusted to 7.3 with KOH. Data were obtained with an Axopatch 200B (Axon Instruments, Union City, Calif., USA), filtered at 2 kHz and digitized at 10 kHz, acquired and analyzed with pClamp9 (Axon Instruments, Union City, Calif., USA). Values are expressed as mean±sem. For statistical comparisons we used the Kolmogorov-Smirnov test. The level of significance was set at P=0.05.
Physical and Behavioral Properties of TAAR1LacZ/LaCZ Mice
The general health, physical state and sensory functions of the TAAR1LacZ/LaCZ mouse line was examined according to a modified version of standard procedures used for behavioral phenotyping of genetically modified mice (Irwin, S. Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia 13, 222-257 (1968); Hatcher, J. P. et al. Development of SHIRPA to characterise the phenotype of gene-targeted mice. Behav. Brain Res. 125, 43-47 (2001)). The comparison of TAAR1+/LacZ and TAAR1LacZ/LacZ mice to their wild-type siblings did not reveal any significant differences regarding their general state of health, their viability, fertility, lifespan, nest building behaviour (
TAAR1LacZ/LacZ Mice Display Elevated Sensitivity to Psychostimulants
Recent observations indicate that at least part of the pharmacological effects of trace amines are due to modulation of catecholamine neurotransmission (Berry, M. D. Mammalian central nervous system trace amines. Pharmacologic amphetamines, physiologic neuromodulators. J. Neurochem. 90, 275-271 (2004); Geracitano, R., Federici, M., Prisco, S., Bernardi, G. & Mercuri N. B. Inhibitory effects of trace amines on rat midbrain dopaminergic neurons. Neuropharmacol. 46, 807-814 (2004)). In addition, TAAR1 has been found to be localized in brain areas with pronounced dopaminergic and serotonergic neurotransmission (Borowsky, B. et al. Trace amines: identification of a family of mammalian G protein-coupled receptors. Proc. Natl. Acad. Sci. USA 98, 8966-8971 (2001)). Amphetamines are known to act as indirect catecholamine agonists that achieve their pharmacological effects by inducing the release of cytosolic dopamine and norephinephrine (King, G. R. & Ellinwood, E. H. Amphetamines and other stimulants. In Lowinson, J. H., Ruiz, P., Millman, R. B. & Langrod, J. G., editors. Substance abuse: a comprehensive textbook, Williams & Wilkins., Baltimore, 1992). Increased extracellular levels of these neurotransmitters, in turn, produce hyperlocomotor activity. The effect of d-amphetamine (2.5 mg/kg i.p.) on locomotor function was therefore compared between TAAR1LacZ/LacZ and TAAR1+/+ mice. Whereas the locomotor activity decreased in wild type mice after d-amphetamine injection regarding total distance moved TAAR1LacZ/LacZ mice were first more active and then moved significantly more than wild type littermates (
The behavioral changes were further investigated in microdialysis studies. The effect of d-amphetamine on the extracellular levels of catecholamines in the striatum revealed 2.3 fold increased levels of dopamine and norepinephrine in TAAAR1LacZ/LacZ compared to wild type mice (
TAAR1 Activity Decreases the Spontaneous Firing Rate of Dopaminergic Neurons in the VTA
The spontaneous firing rate of dopaminergic neurons in the VTA was determined under current clamp conditions. The mean spike frequency in TAAR1+/+ (n=22) and in TAAR1LacZ/LacZ (n=25) was 2.3±0.8 Hz and 17.2±1.2 Hz (p<0.0001,
| Number | Date | Country | Kind |
|---|---|---|---|
| 05108145.3 | Sep 2005 | EP | regional |
