Methods for creating synthetic chromosomes having gene regulatory systems and uses thereof

A technology for chromosome regulation and control, applied in biochemical equipment and methods, other methods for inserting foreign genetic materials, stably introducing foreign DNA into chromosomes, etc., can solve problems that cannot constitute prior art

Pending Publication Date: 2019-03-01
SYNPLOID BIOTEK LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The applicant expressly reserves the right to demonstrate, where appropriate, that the textually cited articles and methods do not constitute prior art under applicable statutory provisions

Method used

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  • Methods for creating synthetic chromosomes having gene regulatory systems and uses thereof
  • Methods for creating synthetic chromosomes having gene regulatory systems and uses thereof
  • Methods for creating synthetic chromosomes having gene regulatory systems and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: De Novo Generation of Satellite DNA-Based Artificial Chromosomes

[0082] For de novo generation of synthetic chromosomes, exogenous DNA sequences were introduced into the HT1080 synthetic chromosome producing cell line, and upon integration into the interarm heterochromatin region of acrocentromeric chromosomes, shorting of acrocentromeric chromosomes was triggered. Large-scale amplification of arms (rDNA / centromeric regions). During the amplification event, the centromere is duplicated, producing a dicentric chromosome with two active centromeres. Subsequent mitotic events lead to cleavage and segregation of dicentric chromosomes, resulting in fragments approximately 20-120 Mb in size, consisting mainly of satellite repeats with subdomains of the co-amplified transfected transgene , this subdomain may also contain an amplified rDNA copy. Newly generated synthetic chromosomes were verified by observing fluorescent chromosome painting (or FISH) via endogeno...

Embodiment 2

[0084] Example 2: Generation of TET repressor and Cumate repressor delivery vectors.

[0085] One embodiment of the regulatable promoter system to be incorporated on the synthetic platform chromosome is TET-ON, a doxycycline-inducible expression system (Clontech, Inc.). The pAPP500 vector was used as the backbone delivery vector to insert the TET-ON transcription regulator into the synthetic chromosome. Briefly, the pEF1alpha-Tet3G transcriptional regulator was isolated by restriction digestion (BsrGI and HindlII), then processed to fill in the 5' end, and ligated into pAPP500 digested with EcoRY. The resulting plasmid vector (pAPP510; Figure 6 : In pApp590, the following elements are present: SV40\polyA / signal=SV40polyA; attB, AttL_Zeo=site-specific recombination site; human / EF1α / Pr=promoter; Tet-On / 3G=transcriptional regulator ; f1 origin = origin of replication; ZeoR = zeocin resistance) contains a promoterless zeocin resistance encoded behind the attB recombination site...

Embodiment 3

[0087]Example 3: Loading of TET-ON and Cumate-ON transcriptional regulators onto synthetic chromosomes

[0088] The pAPP510 and pAPP590 vectors were used as delivery vectors to sequentially insert the TET-ON transcriptional regulator and the Cumate-ON transcriptional regulator, respectively, into synthetic chromosomes. On day 0, a recipient cell line (eg, HT1080) containing a synthetic chromosome (hSynC) is seeded at ~4E4 cells / well of a 24-well dish such that the wells are ~70% confluent by day 1. Cells were incubated at 37°C, 5% CO 2 Incubate overnight in appropriate medium (eg, DMEM+10% FC3 for HT1080). On day 1, both the delivery vector (eg, pAPP510) and the plasmid encoding the recombinant protein (eg, pCXLamIntR) were transfected into HT1080 cells following the manufacturer's instructions (Fisher Scientific, Lipofectamine LTX with Plus reagent). Transfections were performed in duplicate to allow comparison of drug selection and direct cell sorting. Dilute Lipofectamin...

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Abstract

The present invention encompasses compositions and methods to allow one to deliver and express multiple genes under the control of multiple gene regulatory components in a recipient cell via a synthetic chromosome. The engineering of synthetic chromosomes to contain multiple gene control units permits the construction of complex biological circuits.

Description

[0001] Cross References to Related Applications [0002] This international PCT patent application claims priority to U.S. Provisional Patent Application No. 62 / 321,720, filed April 12, 2016. [0003] Statement on Government Support [0004] This invention was made with United States Government support under Contract D15PC00008 awarded by DARPA. The US Government has certain rights in this invention. field of invention [0005] The field of the invention encompasses methods that allow one to engineer synthetic chromosomes to deliver more than one gene under the control of multiple gene regulatory components. Background of the invention [0006] In the following discussion, certain articles and methods will be described for background and introductory purposes. Nothing contained herein should be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that textually cited articles and methods do not constitute pri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/90C12N15/63C12N15/85C12N15/87
CPCC12N15/85C12N2830/003C07K16/2803C07K16/3007C07K16/32C07K2317/14C07K2317/622
Inventor 爱德华·珀金斯艾米·格林
Owner SYNPLOID BIOTEK LLC
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