Glucose-dependent insulin-secreting cells transfected with a nucleotide sequence encoding GLP-1
First Claim
1. A method for constructing a glucose dependent insulin-secreting cell, in vitro, the method comprising the steps of:
- providing an insulin-secreting cell, in vitro;
isolating from a proglucagon gene a minigene construct consisting of (a) the coding region for a protein selected from the group consisting of glucagon-like peptide-1 (GLP-1) and an analog of GLP-1, said analog of GLP-1 being further selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 3;
(b) a partial fragment of an IP sequence at the 5′
-end; and
(c) a nucleotide sequence at the 3′
-end wherein the nucleotide sequence at the 3′
-end is selected from the group consisting of, an entire IP-2 sequence and an entire IP-2 sequence along with a partial fragment of a glucagon-like peptide-2 (GLP-2) sequence;
operably linking a promoter to the minigene construct;
inserting or ligating the minigene construct into a plasmid; and
transfecting the plasmid into the insulin-secreting cell,whereby said insulin-secreting cell produces GLP-1 or an analog of GLP-1 in a glucose dependent manner.
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Abstract
Disclosed herein are cells that secrete insulin in a glucose-dependent manner. The cell line comprises insulin-secreting cells that have been transfected with a minigene construct comprising a nucleotide sequence encoding for glucagon-like peptide-1 (GLP-1). In preferred embodiments, the minigene construct is operatively associated with a promoter. The cell line may be used to treat diabetes or other conditions in which delivering insulin in a glucose-dependent manner would be advantageous, to investigate the function and development of pancreatic cells, and to test the efficacy of drugs that stimulate insulin secretion. The cells may be implanted in a mammal, or may be included in a device that resides exterior to the mammal, yet which delivers insulin to the mammal in response to the glucose level of a body fluid in contact therewith. The minigene construct may also be implemented in conjunction with an in vivo gene transfer approach.
81 Citations
24 Claims
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1. A method for constructing a glucose dependent insulin-secreting cell, in vitro, the method comprising the steps of:
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providing an insulin-secreting cell, in vitro; isolating from a proglucagon gene a minigene construct consisting of (a) the coding region for a protein selected from the group consisting of glucagon-like peptide-1 (GLP-1) and an analog of GLP-1, said analog of GLP-1 being further selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 3;
(b) a partial fragment of an IP sequence at the 5′
-end; and
(c) a nucleotide sequence at the 3′
-end wherein the nucleotide sequence at the 3′
-end is selected from the group consisting of, an entire IP-2 sequence and an entire IP-2 sequence along with a partial fragment of a glucagon-like peptide-2 (GLP-2) sequence;operably linking a promoter to the minigene construct; inserting or ligating the minigene construct into a plasmid; and transfecting the plasmid into the insulin-secreting cell, whereby said insulin-secreting cell produces GLP-1 or an analog of GLP-1 in a glucose dependent manner. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method for constructing a glucose-dependent insulin-secreting cell, in vitro, the method comprising the steps of:
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providing an insulin-secreting cell, in vitro; isolating from a proglucagon gene a minigene construct, SEQ ID NO;
6;operably linking a promoter to the minigene construct; inserting or ligating the minigene construct into a plasmid; and transfecting the plasmid into the insulin-secreting cell, whereby said insulin-secreting cell produces GLP-1 or an analog of GLP-1 in a glucose-dependent manner. - View Dependent Claims (9, 10, 11, 12)
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13. An insulin-secreting cell, produced by a method comprising:
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providing an insulin-secreting cell, in vitro; isolating from a proglucagon gene a minigene construct consisting of (a) the coding region for a protein selected from the group consisting of glucagon-like peptide-1 (GLP-1) and an analog of GLP-1, said analog of GLP-1 being further selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 3;
(b) a partial fragment of an IP sequence at the 5′
-end; and
(c) a nucleotide sequence at the 3′
-end wherein the nucleotide sequence at the 3′
-end is selected from the group consisting of, an entire IP-2 sequence and an entire IP-2 sequence along with a partial fragment of a glucagon-like peptide-2 (GLP-2) sequence;operably linking a promoter to the minigene construct; inserting or ligating the minigene construct into a plasmid; and transfecting the plasmid into the insulin-secreting cell, whereby said insulin-secreting cell produces GLP-1 or an analog of GLP-1 in a glucose dependent manner. - View Dependent Claims (14, 15, 16, 17, 18, 19)
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20. An insulin-secreting cell, produced by a method comprising:
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providing an insulin-secreting cell, in vitro; isolating from a proglucagon gene a minigene construct consisting of SEQ ID NO;
6;operably linking a promoter to the minigene construct; inserting or ligating the minigene construct into a plasmid; and transfecting the plasmid into the insulin-secreting cell, whereby said insulin-secreting cell produces GLP-1 or an analog of GLP-1 in a glucose-dependent manner. - View Dependent Claims (21, 22, 23, 24)
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Specification