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Nucleic acids isolated from the intestine

a technology of nucleic acids and intestines, applied in the field of new avian promoters, can solve the problem of modest gene activation by these transcription factors

Inactive Publication Date: 2010-12-09
UNIVERSITY OF CINCINNATI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to novel isolated avian nucleic acids that contain the gene expression control region of the intestinal fatty acid binding protein (iFABP). These nucleic acids can be used to direct the tissue-specific expression of polypeptides in avian cells. The isolated nucleic acids contain gene regulatory elements and stretches of nucleic acid that may organize the gene regulatory elements in an ordered array relative to a polypeptide-encoding region. The nucleic acids can be optimized for codon usage by a host cell and can further include a polyadenylation signal sequence. The invention also includes expression vectors, recombinant cells, tissues, and animals containing non-naturally occurring recombinant nucleic acid molecules according to the present invention. The technical effects of the invention include the ability to direct tissue-specific expression of polypeptides in avian cells and the potential for improved translation initiation and expression of polypeptides in a chicken gut-specific cell.

Problems solved by technology

However, gene activation by these transcription factors is modest, and additional cell-specific factors are probably required for in vivo regulation.

Method used

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  • Nucleic acids isolated from the intestine
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Examples

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example 1

Molecular Cloning of a Chick iFABP cDNA Fragment

[0139]Total RNA was extracted from the small intestine of an adult female chicken (Gallus gallus) by using Tri Reagent™ (Molecular Research Center Inc., Cincinnati, Ohio). The first strand cDNA was obtained by reverse transcription using oligio-deoxythymidine (12-18 mer, Amersham-Pharmacia) as the primer and Superscript™ II reverse transcriptase (GIBCO-BRL). A DNA fragment of the cFABP 2 gene was amplified from the chick genomic DNA by polymerase chain reaction (PCR) using a high fidelity Taq polymerase (ExTae™; Takara Shuzo Co. Ltd., Tokyo) and the primers FABPI-Fw1, 5′-GAGAACTATGAGAAGTTCATGG-3′ (SEQ ID NO: 6); and FABPI-Rv2, 5′-ACTTGAATTTGTTTCCNTCYTG-3′ (SEQ ID NO: 7). The primers were designed by comparing the iFABP cDNA sequences of human (Genbank accession number: M18079), mouse (M65033), rat (J00732) and Xenopus (L19946). The PCR was manually hot-started and performed at 95° C. for 30 secs, 56° C. for 30 secs, 72° C. for 45 secs ...

example 2

Cloning of an Intron of Chick iFABP Gene

[0140]Chicken genomic DNA was obtained from the hepatic tissue of an adult female chicken by an ordinary method. Briefly, tissue pieces were digested with RnaseA and proteinase K, and genomic DNA was purified by phenol / chloroform extraction followed by ethanol precipitation. The PCR primers, specific to the chick iFABP gene and designed according to the sequence of the cDNA, were FABPI-Fw2: 5′-TGAGTACTATGAGAAGTTCATGGAAGCAATG-3′ (SEQ ID NO: 8) and FABPI-Rv2: 5′-TCCTGCAGAATAGTAAGCTTCAGATTATCGTG-3′ (SEQ ID NO: 9). The chicken iFABP gene fragment that contained the first intron was amplified from the chicken genomic DNA by PCR using Takara LA-Taq and by following a standard protocol for the enzyme. As a result, a single-band product of approximately 600 by size was obtained and T / A-cloned into pGEM-T Easy. The nucleic acid sequence was then determined.

example 3

Cloning of the 5′-Flanking Region of Chicken iFABP Gene

[0141]The 5′-flanking region of chicken iFABP gene was amplified by suppression PCR, as described in Diatchenko et al., Methods Enzymol., 303:349-380 (1999), followed by nested PCR. Briefly, chicken genomic DNA was digested by Hind III because Southern blot analysis of genomic DNA using a 5′ fragment of the first intron of the chick iFABP gene indicated that Hind III digestion gave a single hybridizing band of about 2 kb. The Hind III-digested genomic DNA was treated with Klenow fragment to blunt-end the cleaved genomic fragments, and an adapter DNA (the Adapter 1 of the Smart PCR Substraction Kit, Clontech) was ligated to it. After filling the 3′ end of the adapter by ExTaq DNA polymerase at 75° C. for 5 min, the 5′-flanking region of the chick iFABP gene was amplified by suppression PCR using ExTaq polymerase, PCR1 primer: 5′-TAATACGACTCACTATAGGGC-3′ (SEQ ID NO: 10) and the cFABPI-Rv3b primer: 5′-GTGCAAGGGCAAAATAGCAGAC-3′ (SEQ...

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Abstract

A recombinant intestinal nucleic acid is provided. One embodiment of the present invention contemplates the use of a gut-specific promoter, wherein a promoter can be the chicken intestinal fatty acid binding protein promoter region. A method for making a transgenic bird is also disclosed by transfecting a bird with a vector comprising a recombinant nucleic acid comprising a chicken intestinal fatty acid binding protein promoter region operably linked to a heterologous nucleic acid expressing a desired polypeptide to be expressed in the gut tissue of an avian.

Description

RELATED APPLICATION INFORMATION[0001]This application is a continuation of U.S. patent application Ser. No. 11 / 516,357, filed Sep. 6, 2006, the disclosure of which is incorporated in its entirety herein by reference, which is a continuation of U.S. patent application Ser. No. 10 / 099,663, filed Mar. 14, 2002, now U.S. Pat. No. 7,135,562, issued Nov. 14, 2006, the disclosure of which is incorporated in its entirety herein by reference.FIELD OF THE INVENTION[0002]The present invention relates generally to a novel avian promoter that regulates tissue-specific protein expression. More specifically, the invention relates to a promoter that, in avians, regulates gut-specific expression of a nucleotide sequence under the control of the promoters as, for example, a nucleotide sequence that imparts disease resistance.BACKGROUND[0003]Genetic engineering techniques that provide for transferring a foreign, or exogenous, gene into a host's genome resulting in the production of a transgenic animal...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/00C07H21/00C12N15/63A01K67/027C12N5/06C12N9/64
CPCA01K2217/206C12N15/8509A01K2227/30
Inventor HORSEMAN, NELSON D.PRATT, SCOTT L.
Owner UNIVERSITY OF CINCINNATI
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