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Compositions and methods for metabolic selection of transfected cells

A cell and cell sterol technology, which is applied in the field of compositions and methods for metabolic selection of transfected cells, can solve the problems of unstable coupling process, easy occurrence, and difficulty in filtering through small-pore sterile membranes.

Active Publication Date: 2008-06-04
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

[0160] Example 1: Elaboration of Cholesterol Screening of Mouse Myeloma Cell Line NS-0 in Chemically Defined or / and Serum-Free Cell Culture Medium

[0161] The mouse myeloma cell line NS-0 was adapted in two chemically defined serum-free cell culture media to test the suitability of 3-KSR as a selectable marker in this cellular background or under SFM conditions. Using CD-hybridoma ( ) and CDM-4-NS-0 ( ) for adaptive culture of NS-0 cells. NS-0 cell lines from each adapted culture in CD-SFM were used to establish the Accession cell bank. Establish a method that can be used to determine the dependence of CD-SFM-NS-0 cell line on cholesterol in cell culture medium. The results of these experiments indicated that less than 10% of CD-SFM-NS-0 cells could survive after 72 hours of culture in cholesterol-free medium. After an additional 5 days of culture in CD-SFM, no viable cells were detected using trypan blue staining. In addition, in the absence of cholesterol, no intermi...

Embodiment 2

[0162] Embodiment 2: the PCR reaction of mouse 3-ketosterol reductase (3-KSR) and construction 3-KSR expression vector

[0163] PCR amplification of the 3-ketosterol reductase (3-KSR) gene encoding the mouse muscuhis 3B-hydroxy-delta(5)-steroid deseroid was performed using cDNA isolated from adult male BALB / c mouse kidney Hydrogenase (Hsd3b5). Using the mouse 3-KSRHsd3b5 sequence reported in the references, using its specific oligonucleotide sequence for PCR amplification reaction, a 1.1 kb band can be detected in agarose gel electrophoresis. This band was isolated and cloned into the pCR-Blunt II-TOPO vector ( , and then re-cloned into the pBV dhfr.1 plasmid background, the new building block vector is called pBFksr.1.

[0164] The 1.1kb region encoding 3-ksr in pBFksr.1 was confirmed by DNA sequencing, and its open reading frame (orf) was compared with the published sequence, and the sequencing results were consistent with the reported mouse 3-ketosterol reductase ( NCBI...

Embodiment 3

[0165] Example 3: Transfection and selection of NS-0 cells in chemically defined fatty acid-free selection medium

[0166] A pBF / csr.1 containing the correct 3-KSR ORF was transfected into NS-0 cells and selected in chemically defined fatty acid-free selection medium. The cell selection medium includes the following components: CD-hybridoma ( ), Glutamax (2mM, ), NEAA (non-essential amino acids) (Ix, ), fatty acid-free BSA (1%, Calbiochem), recombinant human IL-6 (5ng / ml, Promega), ITS broth supplement (Ix, Sigma-Aldrich). Initial transfection and selection can be performed in T-75 cell culture flasks. On the day of transfection, NS-0 cells were counted using trypan blue staining to differentiate live or dead cells. At least 90% of the cells survived. Approximately 1 x 10 7 . In addition to plasmid transfection, a negative control transfection (containing no DNA) needs to be performed. The cells were centrifuged, resuspended in 20 ml of serum-free transfection mediu...

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Abstract

The present invention relates to novel selection marker vectors, and methods for using these vectors to generate stable gene expression systems in eukaryotic cells utilizing any enzyme useful in the eukaryotic sterol / cholesterol biosynthetic pathway, such as a 3-ketosteroid reductase, as a metabolic selection marker to select transfected cells. In one embodiment, the method comprises transfecting cells that are auxotrophic for cholesterol with a vector encoding 3-ketosteroid reductase and at least one heterologous protein, and selecting cells that have the ability to survive in medium lacking cholesterol and / or producing the heterologous protein in these cells in chemically defined and / or serum-free media.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 60 / 681,969 filed May 18, 2005 under 35.U.S.C. §119(e) of the US Patent Code, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates to novel marker selection vectors and methods for stably expressing gene products in eukaryotic cells using these vectors. Background technique [0004] The development of in vitro culture technology for mammalian cells has revolutionized biological research. Cell culture technology can be used to evaluate the toxicity or effect of drugs in the early stage of development, and the use of human clinical trials in the early stage of drug development has high risks. Cell culture technology can also be used to generate complex human proteins such as monoclonal antibodies for therapeutic use, and can also be used as a platform for cell-based therapy. These cell c...

Claims

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Application Information

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IPC IPC(8): C08B1/02
CPCC12Y101/0127C12N9/0006C12N15/09C12N15/64
Inventor 路易斯·布兰科达里尔·B·桑佩
Owner BIOFACTURA INC
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