Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1992-05-08
2003-08-05
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S071200, C435S252300, C435S254110, C435S252330, C435S320100, C435S471000, C435S325000, C536S023100, C536S023510, C530S399000
Reexamination Certificate
active
06602687
ABSTRACT:
1. Introduction
2. Background of the Invention
2.1. Biology of Neurotrophic Factors
2.2. Ciliary Neurotrophic Factor
2.3. Functional Properties of Ciliary Neurotrophic Factor
3. Summary of the Invention
3.1. Abbreviations
4. Description of the Figures
5. Detailed Description of the Invention
5.1. Purification of CNTF
5.2. CNTF Bioassays
5.3. Sequencing of CNTF Protein
5.4. Cloning of CNTF-Encoding DNA
5.5. Expression of a CNTF Gene
5.5.1. Identification and Purification of the Expressed Gene Product
5.6. CNTF Genes and Proteins
5.7. Generation of Anti-CNTF Antibodies
5.8. Utility of the Invention
5.8.1. Diagnostic Applications
5.8.2. Therapeutic Applications
5.8.3. Pharmaceutical compositions
5.8.4. Molecular Probes of the Invention May Be Used to Identify Novel CNTF-Homologous Molecules
6. Example: Molecular Cloning, Expression and Regional Distribution of Rat Ciliary Neurotrophic Factor (CNTF)
6.1. Materials and Methods
6.1.1. Purification and Cleavage of CNTF
6.1.2. Generation of cDNA CNTF Clones
6.1.3. Northern Blot
6.1.4. Expression Of Recombinant CNTF
6.2. Results
6.2.1. Determination of CNTF Amino Acid Sequence
6.2.2. Generation of CNTF cDNA Clones and Sequence Analysis
6.2.3. Expression of Recombinant CNTF
6.2.4. Northern Blot Analysis
6.3. Discussion
7. Example: Expression of CNTF In
Escherichia Coli
7.1. Materials and Methods
7.1.1 Construction of a CNTF Expression Vector
7.1.2 Identification of Bacteria Containing the CNTF Expression Vector
7.2. Results and Discussion
8. Example: Cloning of the Human CNTF Gene
8.1. Materials and Methods
8.1.1. DNA, Plasmid and Phage Vectors
8.1.2. Polymerase Chain Reaction
8.2. Results and Discussion
8.2.1. Evidence for the Existence of a Human CNTF Gene
8.2.2. Cloning of a Fragment of the Human CNTF Gene Amplified by PCR
8.2.3. Cloning of the Human CNTF Gene from a Genomic Library
9. Example: Utility of CNTF-Derived Peptide Fragments
9.1. Materials and Methods
9.1.1. Synthesis of Peptides
9.1.2. Cell Culture
9.1.3. Immunization Protocol
9.2. Results and Discussion
9.2.1. Ability of Antibodies Directed Toward a Synthetic Peptide to Neutralize CNTF Activity
9.2.2. Neurotrophic Activity of a Synthetic CNTF Peptide Fragment
9.2.3. Ability Of Antibodies Directed Toward A Synthetic Peptide To Identify CNTF Containing
10. Example: Ciliary Neurotrophic Factor Promotes Survival of Spinal Cord Neurons
10.1. Materials and Methods
10.1.1. Experimental Animals
10.1.2. Tissue Culture Techniques
10.2. Results and Discussion
10.2.1. Effects of Ciliary Neurotrophic Factor (CNTF) on Mediodorsal (MD) Spinal Cord Neurons
10.2.2. Effects of CNTF on Ventral Spinal Cord Neurons
11. Example: Purified Rat Sciatic Nerve CNTF Prevents Lesion-Induced Cell Death of Motorneurons in the Facial Nerve (VIIth Cranial Nerve) of the Newborn Rat
11.1. Materials And Methods
11.2. Results And Discussion
12. Example: High Level Expression And Purification Of Recombinant Human And Rat Ciliary Neurotrophic Factors In
Escherichia Coli
12.1. Materials And Methods
12.1.1. Bacterial Strains And Plasmids
12.1.1.1. Rat CNTF Vectors
12.1.1.1.1. pRPN11
12.1.1.1.2. pRPN12
12.1.1.3. pRPN37
12.1.1.1.4. pRPN38
12.1.1.2. Human CNTR Vectors
12.1.1.2.1. pRPN32
12
.
1
.
1
.
2
.
2
. pRPN33, PRPN39 pRPN40
12.1.2. Induction Of Protein Synthesis
12.1.3. “RAPID” Protein Extraction
12.1.4. Chromatography
12.1.5. Peptide Analysis
12.1.5.1. Rat CNTF
12.1.5.2. Human CNTF
12.1.6. Biological Activity
12.1.7. Other Methods
12.2. Results And Discussion
12.2.1. Expression Of Rat CNTF
12.2.1.1. Effect Of Copy Number
12.2.1.2. Effect Of Antibiotic Resistance
12.2.2. Expression Of Human CNTF
12.2.3. Purification Of Rat And Human CNTF
12.2.3.1. Yield
12.2.3.2. Characterization
12.2.4. Biological Activity
13. Example: Effects Of Modified And Truncated Ciliary Neurotrophic Factor Protein On Biological Activity
13.1. Materials And Methods
13.1.1. Construction Of Parental Expression Vectors
13.1.2. Construction Of Modified Human Ciliary Neurotrophic Factor Vectors
13.1.3. Construction Of Modified Rat Ciliary Neurotrophic Factor Vectors
13.1.4. Biological Assay Of Ciliary Neurotrophic Factor Activity
13.2. Results And Discussion
14. Example: Additional Effects Of CNTF On Ventral Spinal Cord Neurons
14.1. Materials And Methods
14.1.1. Experimental Animals
14.1.2. Tissue Culture Techniques
14.1.3. Neurofilament (NF) Assay
14.1.4. Choline Acetytransferease (CAT) Assay
14.1.5. Histochemical Staining For Acetylcholinesterase (AchE)
14.1.6. Fractionation Of Ventral Horn Cells by Metrizamide Density Gradient
14.2. Results And Discussion
14.2.1. General Morphologies Of Cultures
14.2.2. Effects Of CNTF On Neurofilament (NF) Levels
14.2.3. Effects Of CNTF On Survival Of AChE-Containing Neurons
14.2.4. Effects Of CNTF In Cat Activity
14.2.5. Delayed Addition Experiment
14.2.6. Effects Of CNTF On Ventral Horn Cultures In The Absence Of Glia
14.2.7. Effects Of CNTF On Metrizamide Gradient-Purified Motorneurons
15. Example: Effect Of Ciliary Neurotrophic Factor On Hippocampal Cultures
15.1. Materials And Methods
15.1.1. Hippocampal Cell Cultures
15.1.2. Assay For GAD Enzyme Activity
15.1.3. Measurement Of Neurofilament Protein
15.1.4. Measurement Of High Affinity GABA Uptake
15.1.5. Immunohistochemical Staining For GAD Or GABA
15.1.6. Immunohistochemical Staining For Neuron-Specific Enolase (NSE)
15.1.7. Histochemical Staining For Calbindin
15.1.8. Histochemical Staining For Acetylcholinesterase
15.1.9. Ciliary Neurotrophic Factor
15.2. Results
15.3. Discussion
16. Example: Novel Monoclonal Antibodies To Ciliary Neurotrophic Factor And A Two-Antibody Sandwich Assay For Human Ciliary Neurotrophic Factor
16.1. Materials And Methods
16.1.1. Generation Of Monoclonal Antibodies To Ciliary Neurotrophic Factor
16.1.1.1. Immunization Protocol
16.1.1.2. Hybridoma Formations
16.1.1.3. Screening Of Hybridomas For CNTF Reactivity
16.1.2. Preparation Of Variants Of Human CNTF
16.1.3. Methodology For Two-Site Immunoassay
16.2. Result And Discussion
17. Ciliary Neurotrophic Factor Promotes Survival Of Spinal Motorneurons In Culture
17.1 Material And Methods
17.1.1. Tissue Culture Techniques
17.1.2. Retrograde Labeling Of Motorneurons And Estimation Of The Purity Of The Culture Of Motorneurons
17.2. Results And Discussion
17.2.1. Effect of Ciliary Neurotrophic Factor (CNTF) On Chick Embryonic Spinal Motorneurons In Culture
17.2.2. Survival Effects of Specific Neurotrophic Molecules and Cytokines
17.2.3. Combination of CNTF, Basic FGF and IGF-I
18. Deposit of Microorganism
1. INTRODUCTION
The present invention relates to recombinant DNA molecules encoding ciliary neurotrophic factor (CNTF), and to peptides and proteins derived therefrom. The CNTF and related molecules produced according to the invention may be used to treat a variety of neurological disorders.
2. BACKGROUND OF THE INVENTION
2.1. Biology of Neurotrophic Factors
A number of factors have been identified which influence growth and development in the nervous system. It is believed that these factors may play an important role in sustaining the survival of neuronal populations in the mature, as well as the immature nervous system.
During the normal development of many neuronal populations, there is a defined period of cell death in which many members of the original population die (Hamburger and Levi-Montalcini, 1949, J. Exp. Zool. III:457-501; Hamburger, 1958, Amer. J. Anat. 102:365:410; Hamburger, 1975, J. Comp. Neurol. 160:535-546; Cowan and Wenger, 1968, Z. Exp. Zool., 168:105-124; Rogers and Cowan, 1973, J. Comp. Neurol. 147:291-320; Clarke and Cowan, 1976, J. Comp. Neurol. 167:143-164; Clarke et al., 1976, J. Comp. Neurol. 167:125-142; Hollyday and Hamburger, 1976, J. Comp. Neurol. 170:311-320; Varon and Bunge, 1978, Annu. Rev. Neurosci. 1:327-362; Cowan et al., 1984, Science 225:1258-1265). Neuronal survival has been shown to be proportional to the size of the territory innervated; the smaller the target area of a given neuronal population, the fewer the number of neurons which will survive the period of cell death. It has been suggested that the amount of neurotrophic
Arakawa Yoshihiro
Carroll Patrick Desmond
Furth Mark E.
Gotz Rudolf Georg
Ip Nancy
Mertz Prema
Pennie & Edmonds LLP
Regeneron Pharmaceuticals Inc.
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