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US-12624073-B2 - Specific nuclear-anchored independent labeling system

US12624073B2US 12624073 B2US12624073 B2US 12624073B2US-12624073-B2

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.

Inventors

  • Andreas R. Pfenning
  • Easwaran Ramamurthy
  • Alyssa Lawler

Assignees

  • CARNEGIE MELLON UNIVERSITY

Dates

Publication Date
20260512
Application Date
20200618

Claims (20)

  1. 1 . A nucleic acid construct comprising: (a) a nucleotide sequence encoding a tagged Sun1 fusion polypeptide, wherein the tagged Sun1 fusion polypeptide comprises (i) an N-truncated fragment of a Sun1 polypeptide that is 400 to 600 amino acids in length, and (ii) a tag polypeptide, and (b) a promoter sequence specific for a selected neuronal cell type, wherein the selected neuronal cell type is parvalbumin positive (PV+) neurons, wherein the promoter sequence is operably linked to the nucleotide sequence encoding the tagged Sun1 fusion polypeptide and is effective to drive expression of the sequence encoding the tagged Sun1 fusion polypeptide in the selected cell type, and wherein said tagged Sun1 fusion polypeptide localizes to the nuclear membrane after said expression.
  2. 2 . The nucleic acid construct of claim 1 , wherein the N-truncated fragment of the Sun1 polypeptide has at least 95% sequence identity to amino acids 208-757 of the mouse Sun1 protein having the amino acid sequence set forth in SEQ ID NO: 1.
  3. 3 . The nucleic acid construct of claim 1 , wherein the tag polypeptide is a fluorescent polypeptide.
  4. 4 . The nucleic acid construct of claim 3 , wherein the fluorescent polypeptide is a green fluorescent protein (GFP).
  5. 5 . The nucleic acid construct of claim 4 , wherein the amino acid sequence of the GFP is at least 95% identical to the superfolder GFP sequence set forth in SEQ ID NO:2.
  6. 6 . The nucleic acid construct of claim 1 , wherein the construct further comprises a virus sequence.
  7. 7 . The nucleic acid construct of claim 6 , wherein the virus sequence is an adeno-associated virus (AAV) sequence or a lentivirus sequence.
  8. 8 . A virus particle comprising the nucleic acid construct of claim 1 .
  9. 9 . The virus particle of claim 8 , wherein the virus is AAV or lentivirus.
  10. 10 . A nucleic acid construct comprising: (a) a nucleotide sequence encoding a tagged Sun1 fusion polypeptide, wherein the tagged Sun1 fusion polypeptide comprises (i) an N-truncated fragment of a Sun1 polypeptide that is 400 to 600 amino acids in length, and (ii) a sequence encoding a tag polypeptide, (b) a first lox sequence flanking the 5′ end of the sequence encoding the tagged Sun 1 fusion polypeptide and a second lox sequence flanking the 3′ end of the sequence encoding the tagged Sun1 fusion polypeptide, and (c) a promoter sequence specific for a selected neuronal cell type downstream of the lox sequence flanking the 3′ end of the sequence encoding the tagged Sun1 fusion polypeptide, wherein the selected neuronal cell type is parvalbumin positive (PV+) neurons, such that the sequence encoding the tagged Sun1 fusion polypeptide is in reverse orientation with respect to the promoter, wherein said tagged Sun1 fusion polypeptide localizes to the nuclear membrane after being expressed.
  11. 11 . The nucleic acid construct of claim 10 , wherein the N-truncated Sun1 polypeptide has at least 95% sequence identity to amino acids 208-757 of the mouse Sun1 protein having the amino acid sequence set forth in SEQ ID NO:1.
  12. 12 . The nucleic acid construct of claim 10 , wherein the tag polypeptide is a fluorescent polypeptide.
  13. 13 . The nucleic acid construct of claim 12 , wherein the fluorescent polypeptide is a GFP.
  14. 14 . The nucleic acid construct of claim 13 , wherein the amino acid sequence of the GFP is at least 95% identical to the superfolder GFP sequence set forth in SEQ ID NO:2.
  15. 15 . The nucleic acid construct of claim 10 , wherein the construct further comprises a virus sequence.
  16. 16 . The nucleic acid construct of claim 15 , wherein the virus sequence is an AAV sequence or a lentivirus sequence.
  17. 17 . A virus particle comprising the nucleic acid construct of claim 10 .
  18. 18 . The virus particle of claim 17 , wherein the virus is AAV or lentivirus.
  19. 19 . A method for labeling a selected neuronal cell type within a population of different cell types, comprising introducing into the population of different cell types a nucleic acid construct comprising: (a) a nucleotide sequence encoding a tagged Sun1 fusion polypeptide, wherein the tagged Sun1 fusion polypeptide comprises (i) an N-truncated fragment of a Sun1 polypeptide that is 400 to 600 amino acids in length, and (ii) a sequence encoding a tag polypeptide, and (b) a promoter sequence specific for the selected neuronal cell type, wherein the selected neuronal cell type is parvalbumin positive (PV+) neurons, wherein the promoter sequence is operably linked to the sequence encoding the tagged Sun1 fusion polypeptide, and is effective to drive expression of the sequence encoding the tagged Sun1 fusion polypeptide in the selected cell type, and 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.
  20. 20 . The method of claim 19 , wherein the N-truncated fragment of the Sun1 polypeptide has at least 95% sequence identity to amino acids 208-757 of the mouse Sun1 protein having the amino acid sequence set forth in SEQ ID NO: 1.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a U.S. National Stage Application under 35 U.S.C. § 371 of International Application Serial No. PCT/US2020/038520, filed on Jun. 18, 2020, which claims benefit of priority from U.S. Provisional Application Ser. No. 62/921,452, filed Jun. 18, 2019. The disclosure of each of the prior applications is considered part of (and is incorporated by reference in) the disclosure of this application. TECHNICAL FIELD This document relates to materials and methods for labeling and subsequently isolating particular cell types from mixed cell populations. BACKGROUND The brain and other tissues consist of heterogeneous populations of cells with specialized properties and functions. Genetically and epigenetically distinguishable cell types are likely to play different roles and exhibit different responses in disease and other conditions. The vast majority of genomic assays, however, are conducted on bulk tissue and not individual cell types, making the results of such assays difficult to interpret and disentangle for cell type-specific mechanisms. Improved techniques for nuclei labeling and isolation are needed to solve this problem. SUMMARY This document is based, at least in part, on the development of compositions and methods for labeling and isolating specific populations of cells. The methods are referred to herein as SNAIL (Specific Nuclear-Anchored Independent Labeling) and cSNAIL (Cre-Specific Nuclear-Anchored Independent Labeling). cSNAIL and SNAIL provide improvements over current methods for nuclear isolation methods, as they are easier, more time-efficient, and more cost-effective, without any loss of selection. Moreover, cSNAIL and SNAIL have the added benefits of being compatible with multiplexing and other transgenic models, extending to new cell types, and being transferrable across species. The availability of this technology increases the practicality of cell type-specific genomics, and allows these approaches to be used in other mammals, including humans. In a first aspect, this document features a nucleic acid construct containing (a) a sequence encoding a tagged Sun1 fusion polypeptide, where the tagged Sun1 fusion polypeptide comprises, consists of, or consists essentially of (i) an N-truncated fragment of a Sun1 gene, where the fragment encodes at least a portion of a portion of a Sun1 protein, and where the portion of the Sun1 protein is 400 to 600 amino acids in length, and (ii) a sequence encoding a tag polypeptide, and (b) a promoter sequence specific for a selected cell type, where the promoter sequence is operably linked to the sequence encoding the tagged Sun1 fusion polypeptide, and is effective to drive expression of the sequence encoding the tagged Sun1 fusion polypeptide in the selected cell type. (By “consists essentially of” is meant that a nucleic acid or a polypeptide contains specified components, and can contain additional sequences that do not materially affect the basic and novel characteristics of the nucleic acid or polypeptide.) The N-truncated fragment of the Sun1 gene can encode a polypeptide with at least 95% sequence identity to amino acids 208-757 of the mouse Sun1 protein having the amino acid sequence set forth in SEQ ID NO:1. The tag polypeptide can be a fluorescent polypeptide (e.g., a green fluorescent protein (GFP), such as a GFP with an amino acid sequence that at least 95% identical to the superfolder GFP sequence set forth in SEQ ID NO:2). The construct can further include virus sequences (e.g., adeno-associated virus (AAV) sequences or lentivirus sequences). The promoter sequence can be specific for parvalbumin positive (PV+) neurons, such the sequence encoding the tagged Sun1 fusion polypeptide is expressed in PV+ neurons but not expressed in at least 90% of parvalbumin negative (PV−) neurons. In another aspect, this document features a nucleic acid construct containing (a) a sequence encoding a tagged Sun1 fusion polypeptide, where the tagged Sun1 fusion polypeptide comprises, consists of, or consists essentially of (i) an N-truncated fragment of a Sun1 gene, where the fragment encodes a portion of a Sun1 protein, and where the portion of the Sun1 protein is 400 to 600 amino acids in length, and (ii) a sequence encoding a tag polypeptide, (b) a first lox sequence flanking the 5′ end of the sequence encoding the tagged Sun1 fusion polypeptide and a second lox sequence flanking the 3′ end of the sequence encoding the tagged Sun1 fusion polypeptide, and (c) a promoter sequence dowvnstream of the lox sequence flanking the 3′ end of the sequence encoding the tagged Sun1 fusion polypeptide, such that the sequence encoding the tagged Sun1 fusion polypeptide is in reverse orientation with respect to the promoter. The N-truncated fragment of the Sun1 gene can encode a polypeptide with at least 95% sequence identity to amino acids 208-757 of the mouse Sun1 protein having the amino acid sequence set forth in SEQ ID