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Host cell modification with artificial endosymbionts

An artificial endosymbiont and host cell technology, applied in the direction of genetically modified cells, fused cells, cells modified by introducing foreign genetic material, etc., can solve the problems of high complexity and complexity of eukaryotic cells, slow growth of bacterial cells, etc. question

Inactive Publication Date: 2016-11-23
BELL BIOSYST
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some eukaryotic cells targeted for recombinant DNA manipulation, e.g., mammalian cells, grow more slowly than bacterial cells, making the selection of eukaryotic cells containing heterologous nucleic acids more time-consuming
The generation of eukaryotic cells containing longer-lasting changes, including heritable changes, often requires the use of homologous recombination, which, while technically advanced, has historically been inefficient
In addition, the introduction of many different heterologous nucleic acids is due to the higher complexity of eukaryotic cells (such as the presence of organelles), the greater degree of unpredictability in response to multiple heterologous factors, and the selection of genes / expressions with multiple genes / expressions. Complicated by technical deficiencies in eukaryotic cells that vary in type

Method used

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  • Host cell modification with artificial endosymbionts
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  • Host cell modification with artificial endosymbionts

Examples

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example 1

[0151] Example 1: Translocation of proteins from artificial endosymbionts into host cells using the type I secretion system

[0152] The type I secretion system (T1SS) in Gram-negative bacteria can be used to export a variety of proteins (their cognate substrates) with different sizes and diverse functions. The MTB genome encodes the T1SS gene (Matsunaga, T. et al., "Completegenome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. AMB-1", DNA Res. 12:157-66, 2005; incorporated herein by reference in its entirety ).

[0153] The target protein, green fluorescent protein (GFP), or red fluorescent protein (RFP), was N-terminally fused to YP_420640 (RTX toxin and related Ca 2+ binding protein), YP_423419 (RTX toxin and related Ca 2+ binding protein), YP_422785 (amb3422), or the C-terminal 200 amino acids of other MTB T1SS substrates. Alternatively, the proteins in MTB-YP_420502, YP_420631.1, YP_420638.1, YP_420640.1, YP_421364.1, YP_422662.1, YP...

example 2

[0159] Example 2: Translocation of proteins from artificial endosymbionts into host cells using the type IV secretion system

[0160] The CagA protein was engineered by replacing the last 20 amino acids with 24 amino acids from the C-terminal end of the RSF1010MobA protein (residues 684-709), which is secreted by the T4SS system. Translocation of CagA-MobA into host cells was monitored by observing the hummingbird phenotype caused by CagA in the host cells. The hummingbird phenotype is disclosed in Segal et al. by the spreading and elongated growth of cells, the presence of lamellipodia (thin sheets of actin present at cell edges), and filopodia-like structures (containing actin Finger-like projections characterize tight bundles of protein filaments (see Segal, E.D. et al., "Altered States: Involvement of Phosphorylated Caga in the Induction of Host Cellular Growth Changes by Helicobacter Pylori", Proc Natl Acad Sci. USA 96(25):14559 -64, 1999).

[0161] The hummingbird phen...

example 3

[0168] Example 3: Translocation of Nucleic Acids from Artificial Endosymbionts into Host Cells Using the Type IV Secretion System

[0169] The CagA4-type secretion system (T4SS) can be used to transfer plasmid DNA from artificial endosymbionts into mammalian host cells. Sequence analysis revealed that components of the T4SS system are present in MTB.

[0170] Plasmid pBBR-MSC was engineered to contain (oriT+trwABC) for plasmid transfer via the T4SS system, and the plasmid was engineered to contain expression of the target protein encoded under the control of the HCMVIE1 promoter-enhancer-first intron box. The target protein is a selectable marker such as DHFR or glutamate synthetase, or a reporter protein such as GFP, or a transcription factor such as cMyc.

[0171] Mammalian selectable markers can be used as target genes. These genes include the puromycin N-acetyltransferase gene for puromycin resistance; blasticidin S deaminase for blasticidin S resistance; and the amino ...

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Abstract

The present invention is directed generally to host cells with artificial endosymbionts, wherein the artificial endosymbiont and the host cell communicate with each other to alter a phenotype of the host cell. In some embodiments, the communication comprises the secretion of a polypeptide from the artificial endosymbiont into the host cell. The secreted polypeptide can be a selectable marker, a reporter protein, a transcription factor, a signal pathway protein, a receptor, a growth factor, a cytokine, an effector molecule or other factors that can produce a phenotype in the host cell.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 61 / 873,308, filed September 3, 2013, the contents of which are hereby incorporated by reference. [0003] Sequence listings, tables or computer programs involved [0004] The official copy of the sequence listing is submitted as an ASCII format text file through EFS-Web at the same time as the instructions, the file name is "BELL.018_ST25.txt", the creation date is September 3, 2014, and the size is 962 kilobytes. The sequence listing submitted via EFS-Web is part of the specification and is hereby incorporated by reference in its entirety. technical field [0005] The present invention generally relates to host cells with artificial endosymbionts. The artificial endosymbiont of the host cell communicates with and alters various aspects of the host cell. Background technique [0006] The advent of recombinant DNA technology created the ability...

Claims

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Application Information

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IPC IPC(8): C12N15/03
CPCC12N2510/00C12N15/87C07K2319/034C07K14/435C12N1/20C12N5/16C07K14/195
Inventor C·B·贝尔A·巴扎罗夫A·瓦克尔J·巴罗佐
Owner BELL BIOSYST
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