Metl1ods'in.Molecular Biology o 8'
`
`Practical
`
`Molecular Virology
`
`Viral Vectors for Gene Expression
`
`Edited by
`
`Mary K. L. Collins
`
`Chester Beatty Laboratories, Institute of Cancer Research,
`London, UK
`
`\ /
`‘j, C" Humana Press 0 Clifton, New Jersey
`
`MERCK v. GENENTECH
`MERCK V. GENENTECH
`IPR2016-01373
`IPR2016-01373
`GENENTECH 2031
`GENENTECH 2031
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`CHAPTER 5
`
`Selectable Markers
`
`for Eukaryotic Cells
`
`Richard Vile
`
`1. Introduction
`
`The transfer of DNA sequences into a population of cells can rarely, if
`ever, be achieved with 100% efliciency. Typically, transfection of cells with the
`calcium phosphate method will transduce only between 0.1 and 1% of the
`cells with the sequences of interest (1), although some workers have achieved
`higher efficiencies (2). If the transferred sequences do not confer a selective
`growth advantage, it is essential to use selection for transduced cells. The
`marker gene itselfmay be the gene of interest, e.g., to label a certain cellular
`population; alternatively, expression of the marker may merely be a convenient
`selection for the cellular population expressing another gene that has been
`cotransfected with the marker. Critically, selectable markers permit positive
`selection (i.e., the cells ofinterest are not killed); this is in contrast to systems in
`which demonstration of infection with a virus leads to death of the recipient
`cell (such as VSV pseudotypes, see Chapter 9).
`
`1.1 . Types ofSelectable Markers
`1.1.1. Dominant Selectable Markers
`
`Many dominant selectable markers are bacterial genes that can be ex-
`pressed in eukaryotic cells when placed under the control of suitable pro-
`moter elements. The recipient cells do not have to posses specific genotypes.
`
`From: Methods in Molecular Biology, Vol. 8:
`Practical Molecular Virology: Viral Vectors for Gene Expression
`Edited by: M. Collins © 1991 The Humana Press Inc., Clifton. NJ
`
`49
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`56
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`-
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`Vile
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`24-multiwell plate, to grow in a range of concentrations of the drug. (For
`cells in suspension, 10‘ cells in 100 },lL should be plated in 96-well plates.)
`The optimal concentration to be used for selection is generally determined
`to be the lowest concentration that kills all the cells within 10-14 days, although
`some antibiotics can kill cells faster than this, so the dying cells should be
`continually monitored. The selective medium should be changed every 2-3
`days during this period to remove dead and dying cells. As stated earlier, it is
`not possible to quote definitive concentrations for any selection system, since
`this depends on the cell type used, the construct from which the marker is
`expressed, and the growth conditions.
`
`.
`
`3.2. Time-Course
`
`for DNA Zhansfer and Subsequent Selection
`
`A typical experimental protocol for the transformation of adherent test
`cells to antibiotic resistance is described below.
`
`1. Plate 5 X 105 cells in a 90—mm plate.
`2. After 16-24 h, transfer DNA into the cells using the method of choice
`(calcium phosphate precipitation, lipofection, electroporation, DEAE
`dextran, or viral infection). The marker gene is included either by itself
`(~15 ug), as cotransfected plasmid (~0.5 pg), or as an integral compo-
`nent of the test DNA sequences.
`3. Leave the transformed cells at least 36-48 h to grow in normal medium.
`This allows the integration and expression of the transferred sequences,
`and the accumulation of the enzymes required to degrade the antibiotic.
`4. Split the cells into the appropriate selection medium. The strength of
`the split depends on the efficiency of the transfer process and, also on
`the optimal cell density requirements for the selection. Typically, 5 x 10‘
`cells are plated into selection medium per 90-mm plate.
`5. Grow the selecting cells for up to 14 d by removing dying cells every 341 ~
`(1 and renewing the selection medium. Separate colonies should become
`visible on the bottom of the plate, which can be cloned using cloning
`rings and expanded for further study.
`
`3.3. Selection for Gene Amplification
`
`Selection for gene amplification frequently requires specific growth media
`for the recipient cells, especially if the amplification is sought after introduc-
`tion of the marker gene into a cell deficient in endogenous activity of the
`gene. Details of these requirements are given in ref. 14. Otherwise, transfec-