Specific nuclear-anchored independent labeling system
Inventors
Pfenning, Andreas R. • Ramamurthy, Easwaran • Lawler, Alyssa
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Assignees
Member
Carnegie Mellon UniversityCarnegie Mellon University is a global research institution based in Pittsburgh, Pennsylvania, recognized for interdisciplinary education, research, and innovation in science, engineering, arts, technology, and social sciences. The university leads advancements in artificial intelligence, robotics, digital health, and performing arts. Located in a technology-driven and culturally rich city, CMU powers real-world impact through research centers, industry engagement, workforce training, and initiatives that shape regional and global communities.
Carnegie Mellon University is a global research institution based in Pittsburgh, Pennsylvania, recognized for interdisciplinary education, research, and innovation in science, engineering, arts, technology, and social sciences. The university leads advancements in artificial intelligence, robotics, digital health, and performing arts. Located in a technology-driven and culturally rich city, CMU powers real-world impact through research centers, industry engagement, workforce training, and initiatives that shape regional and global communities.
Publication Number
US-12624073-B2
Publication Date
2026-05-12
Expiration Date
Abstract
Materials and methods for labeling and isolating particular cell types from mixed cell populations are provided herein. Also provided herein are methods for generating data representing a synthetic genetic sequence configured for labeling at least one cell type by causing expression of a marker in the at least one cell type.
Core Innovation
A nucleic acid construct encodes a tagged Sun1 fusion polypeptide that comprises an N-truncated fragment of a Sun1 polypeptide that is 400 to 600 amino acids in length and a tag polypeptide. The construct further includes a promoter sequence specific for a selected neuronal cell type, wherein the selected neuronal cell type is parvalbumin positive (PV+) neurons, and the promoter drives expression of the tagged Sun1 fusion polypeptide in the selected cell type.
In a PV+ neuron labeling construct, the sequence encoding the tagged Sun1 fusion polypeptide is in reverse orientation with respect to the promoter and is flanked by lox sequences at the 5′ and 3′ ends. In related embodiments, the construct is excised and irreversibly re-inserted into the correct orientation for expression in the selected neuronal cell type by Cre recombinase expressed specifically in PV+ neurons.
The constructs and methods enable labeling of a selected neuronal cell type within a population of different cell types by introducing a nucleic acid construct that drives expression of the tagged Sun1 fusion polypeptide under a cell-type-specific promoter. The tagged Sun1 fusion polypeptide localizes to the nuclear membrane after being expressed. In specified embodiments, the N-truncated Sun1 fragment has at least 95% sequence identity to mouse Sun1 amino acids 208-757, and the tag polypeptide can be a fluorescent polypeptide such as GFP with an amino acid sequence at least 95% identical to the superfolder GFP sequence.
Claims Coverage
The independent claim set includes four independent claims directed to a PV+ neuron-specific nucleic acid construct for nuclear membrane-localized tagged Sun1 labeling, a Cre-dependent reverse-orientation lox-based construct for the same labeling, and methods for labeling PV+ neurons in mixed populations using either direct construct introduction or a two-construct Cre/lox switching arrangement. Each independent claim centers on 2–3 inventive elements: a tagged N-truncated Sun1 fusion, PV+ neuron-specific promoter control, and where applicable reverse orientation with lox sites and Cre excision, together with nuclear membrane localization after expression.
N-truncated Sun1-tag fusion under a PV+ neuron-specific promoter for nuclear membrane localization
A nucleic acid construct comprising a nucleotide sequence encoding a tagged Sun1 fusion polypeptide, wherein the tagged Sun1 fusion polypeptide comprises an N-truncated fragment of a Sun1 polypeptide that is 400 to 600 amino acids in length and a tag polypeptide, and a promoter sequence specific for parvalbumin positive (PV+) neurons, operably linked to the nucleotide sequence and effective to drive expression in the selected cell type, wherein the tagged Sun1 fusion polypeptide localizes to the nuclear membrane after said expression.
Reverse-oriented PV+ neuron nuclear labeling cassette with lox sites for tagged Sun1 expression
A nucleic acid construct comprising a nucleotide sequence encoding a tagged Sun1 fusion polypeptide with an N-truncated Sun1 fragment (400 to 600 amino acids) and a sequence encoding a tag polypeptide; first and second lox sequences flanking the 5′ and 3′ ends of the tagged Sun1 fusion polypeptide sequence; and a promoter sequence specific for PV+ neurons downstream of the lox sequence flanking the 3′ end, wherein the tagged Sun1 fusion polypeptide is in reverse orientation with respect to the promoter, and the tagged Sun1 fusion polypeptide localizes to the nuclear membrane after being expressed.
Method of PV+ neuron labeling by introducing a tagged Sun1 fusion cassette expressed under a PV+ promoter
A method for labeling a selected neuronal cell type within a population of different cell types comprising introducing into the population a nucleic acid construct having a nucleotide sequence encoding a tagged Sun1 fusion polypeptide with an N-truncated Sun1 fragment (400 to 600 amino acids) and a tag polypeptide, and a promoter sequence specific for the selected neuronal cell type (PV+ neurons), operably linked to the fusion and effective to drive expression in the selected cell type, wherein the tagged Sun1 fusion polypeptide is expressed in and thereby labels the selected cell type, and wherein the tagged Sun1 fusion polypeptide localizes to the nuclear membrane after being expressed.
Cre/lox-based irreversibly switched reverse-to-correct orientation PV+ neuron tagged Sun1 labeling
A method for labeling a selected neuronal cell type within a population of different cell types comprising introducing (i) a first nucleic acid construct encoding a tagged Sun1 fusion polypeptide with an N-truncated Sun1 fragment (400 to 600 amino acids) flanked by first and second lox sequences and arranged in reverse orientation with respect to a promoter downstream of the lox sequence flanking the 3′ end, and (ii) a second nucleic acid construct encoding Cre recombinase operably linked to a promoter specific for PV+ neurons; and incubating under conditions in which Cre recombinase is expressed in PV+ neurons, wherein expressed Cre recombinase excises the tagged Sun1 fusion sequence from the nucleic acid construct and irreversibly re-inserts it in the correct orientation for expression in the selected neuronal cell type.
Across the independent claims, the inventive core is a tagged Sun1 fusion polypeptide with an N-truncated Sun1 fragment (400 to 600 amino acids) that localizes to the nuclear membrane when expressed under PV+ neuron-specific promoter control. Additional claim scope is provided by reverse orientation using lox sequences and, in a method, Cre recombinase expressed specifically in PV+ neurons that irreversibly switches the tagged Sun1 cassette into correct orientation for expression.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Labeling a selected neuronal cell type within a population of different cell types, wherein the selected neuronal cell type is parvalbumin positive (PV+) neurons.
Interested in licensing this patent?