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Compositions and methods for metabolic selection of transfected cells

A technology of cells and host cells, applied in the field of compositions and methods for the metabolic selection of transfected cells, capable of solving the inconsistency between batches of cholesterol cell culture fluid preparation, the short half-life of cholesterol growth fluid, and the decrease of cholesterol content, etc. question

Active Publication Date: 2016-11-23
BIOFACTURA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Additionally, the conjugation process is inherently unstable, resulting in a very short half-life of cholesterol-supplemented growth media
Additionally, cholesterol precipitation was more likely to occur when culturing cholesterol-starved cell lines in chemically defined, serum-free cell cultures than in cultures containing fetal bovine serum
Ultimately, cholesterol is not easily filtered through small-pore sterile filters such as PES due to its intrinsic polymer affinity, resulting in a significant decrease in cholesterol levels in the final filtered culture
This phenomenon leads to batch-to-batch inconsistency in the preparation of cell culture media supplemented with cholesterol

Method used

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  • Compositions and methods for metabolic selection of transfected cells
  • Compositions and methods for metabolic selection of transfected cells
  • Compositions and methods for metabolic selection of transfected cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0161] Example 1: Describe the determination of the chemical composition of the mouse myeloma cell line NS-0 or the screening of cholesterol in serum-free cell culture fluid

[0162] The mouse myeloma cell line NS-0 was cultured in two chemically determined serum-free cell culture fluids to test the suitability of 3-KSR as a selection marker in this cell background or SFM conditions. Use CD-hybridoma (Invitrogen ) And CDM-4-NS-0(HyClone ) Adaptation culture of NS-0 cells. Each NS-0 cell line adapted to culture in CD-SFM was used to establish an Accession cell bank. A method was established to determine the dependence of CD-SFM-NS-0 cell line on cholesterol in cell culture fluid. These test results indicate that less than 10% of the CD-SFM-NS-0 cells can survive 72 hours in a cholesterol-free medium. After 5 days of continuous culture in CD-SFM, no viable cells were detected by trypan blue staining. In addition, in the absence of cholesterol, the intermittent growth phase of ...

Embodiment 2

[0163] Example 2: PCR reaction of mouse 3-ketosterol reductase (3-KSR) and construction of 3-KSR expression vector

[0164] The cDNA isolated from the kidney of adult male BALB / c mice was used for PCR amplification of the 3-ketosterol reductase (3-KSR) gene, which encodes mouse muscle 3B-hydroxy-delta(5)-steroid Hydrogenase (Hsd3b5). The mouse 3-KSRHsd3b5 sequence reported in the reference literature was used for PCR amplification with its specific oligonucleotide sequence, and a 1.1kb band could be detected in agarose gel electrophoresis. This band was isolated and cloned into the pCR-Blunt II-TOPO vector (Invitrogen , And then re-cloned into the pBVdhfr.1 plasmid background, the new building block vector is called pBFksr.1.

[0165] The 1.1 kb region encoding 3-ksr in pBFksr.1 was confirmed by DNA sequencing, and its open reading frame (orf) was compared with the published sequence. The sequencing result was consistent with the reported mouse 3-ketosterol reductase ( NCBI regi...

Embodiment 3

[0166] Example 3: Transfection and selection of NS-0 cells in fatty acid-free selective medium with determined chemical composition

[0167] A pBF / csr.1 containing the correct 3-KSR open reading frame was transfected into NS-0 cells and selected in a fatty acid-free selection medium with a defined chemical composition. The cell selection medium includes the following components: CD-hybridoma (Invitrogen ), Glutamax (2mM, Invitrogen ), NEAA (non-essential amino acids) (Ix, Invitrogen ), fatty acid-free BSA (1%, Calbiochem), recombinant human IL-6 (5ng / ml, Promega), ITS liquid culture supplement (Ix, Sigma-Aldrich). The initial transfection and selection can be carried out in T-75 cell culture flasks. On the day of transfection, NS-0 cells were counted by trypan blue staining to distinguish live cells from dead cells. At least 90% of the cells survived. Each transfection requires about 1×10 7 . In addition to plasmid transfection, a negative control transfection (without DNA)...

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Abstract

The present invention relates to novel marker selection vectors and methods of using these vectors to stably express gene products in eukaryotic cells. More specifically, the present invention provides compositions and methods that utilize any enzyme useful in the eukaryotic sterol / cholesterol biosynthesis pathway, such as 3-sterolone reductase as a metabolic selectable marker for selection of transfected cells. In one embodiment, the method comprises transfecting cells that are cholesterol-deficient cells having a vector encoding a 3-sterol reductase and at least one heterologous protein, and selecting selected cells that are viable in a medium lacking cholesterol and and / or intracellular production of heterologous proteins in chemically defined and / or serum-free medium.

Description

[0001] Cross references to related applications [0002] This application claims priority to the U.S. Provisional Application Serial No. 60 / 681,969 filed on May 18, 2005 based on U.S. Patent Law 35.U.S.C.§119(e), which is hereby incorporated by reference in its entirety. Technical field [0003] The present invention relates to new marker selection vectors and methods for using these vectors to stably express gene products in eukaryotic cells. Background technique [0004] The development of in vitro culture technology of mammalian cells is of revolutionary significance to biological research. Cell culture technology can be used to evaluate the toxicity or effect of drugs in the early stage of development. The use of human clinical trials in the early stage of drug development has a high risk. Cell culture technology can also be used to produce complex human proteins such as monoclonal antibodies for therapeutic purposes, and can also be used as a platform for cell-based therapy. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B1/02
CPCC12N9/0006C12Y101/0127C12N15/09C12N15/64
Inventor 路易斯·布兰科达里尔·B·桑佩
Owner BIOFACTURA INC
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