Genetic engineering of animal cells in culture

To study individual genes, it may be necessary to transfer the gene into the cells under study.

In this section we will discuss some of the common methods that are used to introduce

genes into mammalian cells. This is essential so that one can genetically manipulate cells

so that they are able to produce large levels of the desired protein (overexpression).

In the diagram, a gene is spliced into a plasmid, called shuttle vectors, and then introduced

into the mammalian cell.

Outline

1. Transfer of “Naked” DNA 2. Viral assisted DNA transfer3. Selection and Amplification of transfected

cells4. Gene Expression5. Expression cassettes

Lecture 7 Animal Cell BiotechnologyGenetic engineering of animal cells

Mammalian cells are the host cells of choice for the synthesis of complex glycoproteins from recombinant genes

• have required characteristics for post-translational processing and product secretion

• Chinese hamster ovary (CHO) most commonly used, well studied and characterized

– CHO cells are very popular for the expression of human recombinant glycoproteins because the glycosylation enzymes resemble those found in human cell lines. They can grow as anchorage dependent or as cell suspensions.

• Transfection – transfer of nucleic acid into mammalian cells → also refers to DNA-mediated transformation

Lecture 7 Animal Cell BiotechnologyGenetic engineering of animal cells

I. Transfer of ‘naked DNA’

1. Calcium phosphate method

2. DEAE-dextran

3. Encapsulation of DNA in liposome vesicles

4. Electroporation

5. Micro-injection

6. Protoplast fusion

1. Calcium phosphate method

• Discovered in 1973:

– Cheap and simple method

• DNA co-precipitates with calcium phosphate and adsorbs onto the cell membrane (endocytosed)

• Cells are treated with dimethyl sulfoxide (DMSO) or glycerol to allow DNA to pass through the membrane → cell membrane destabilized

• Proportion of cells taking up DNA-precipitate is high, amount incorporated into nucleus is low (1-2%)

2. DEAE-dextran• DNA forms complex with diethylaminoethyl dextran

→ cells treated with DMSO to destabilize membrane

→ high proportion of cells are transfected, but transient protein expression

3. Encapsulation of DNA in liposome vesicles (lipofection)

• transfection with high levels of gene expression complexes of positively charged lipid and DNA

• readily taken up by mammalian cells

• high-efficiency

• Introduction of DNA into cells with commercially available cationic lipids. Charged head groups (positive) are drawn to the phosphate backbone of DNA.

4. Electroporation• Exposure of cell suspension to a high-voltage electrical pulse (100-200 V, 1-2 ms)

• Transient pores are formed in the cell membrane, allows DNA to pass through

• Exposure of suspended cells to a pulsed electric field that causes the transient formation of pores in the cell membrane, allowing for exchange of macromolecules between the extracellular environment and the cytoplasm. Removal of the electrical field results in spontaneous sealing of the pores.

• Microinjection• DNA injected with microneedles or pipettes directly into the nucleus, or into the cytoplasm.

Requires highly trained and skilled personnel. Typically used when there are limited numbers of recipient cells.

http://www.youtube.com/watch?v=s0zM-3dmO4M

http://aditusmedical.com/

5. Micro-injection

• insertion of DNA directly into the nucleus of individual cells

• effective for small numbers of cells, very effective

http://www.youtube.com/watch?v=h-Bfc1GPWpE

6. Protoplast fusion• Bacteria-grown plasmids are introduced

into animal cells via fusion with bacterial protoplasts

• Bacterial cell walls are removed using lysozyme treatment

• Protoplasts fuse to the mammalian cells• DNA purification not required• may introduce bacterial components into

recipient cells, cultures may be overgrown with bacteria

• Bacteria are used to produce plasmids containing the human gene of interest. The cell walls are removed using lysozyme, resulting in the formation of protoplasts. The protoplasts are brought in contact with the mammalian cells, leading to protoplast fusion to mammalian cells, using Polyethylene glycol (PEG).

Lecture 7 Animal Cell BiotechnologyGenetic engineering of animal cells

II. Viral-assisted DNA transfer • animal viruses are very efficient for DNA transfer

(transduction)

→ promote transfer of DNA to cells because of capsid proteins that bind to cell membrane receptors

→ contain promoters that allow expression of inserted genes in animal cells

→ replicate to high copy numbers (several copies of required genes within cell)

→ some viruses’ genomic material integrate into the animal cell genome

Simian virus SV40

• most successful and widely used viral system derived from simian virus SV40

contains circular DNA duplex and contains circular DNA duplex and many well-characterized restriction many well-characterized restriction enzyme cleavage sitesenzyme cleavage sites

part of SV40 genome replaced with part of SV40 genome replaced with foreign DNAforeign DNA

limited host range (only monkey limited host range (only monkey cells)cells)

Retroviruses• Retroviruses are also efficient vectors for

integration of foreign genes into animal cells

• Have RNA genome, complementary DNA copies are formed by the enzyme reverse transcriptase

DNA can be DNA can be incorporated into incorporated into host cells’ genome host cells’ genome without causing cell without causing cell lysislysis

Retroviral Retroviral structural genes are structural genes are replaced with replaced with foreign DNAforeign DNA

Selection and amplification of transfected cells

1. Cell selection by genetic markers

• proportion of cells that accept exogenous DNA may be as low as 1 in 106

• marker gene incorporated into a recipient cell, allows successful transfectants to be identified

• marker can be on the same vector as target gene, or on separate vector

Genetic markers…1. non-dominant genes – selection of mutants that

lack marker enzyme activity prior to transfection

eg. DHFR converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids

Methotrexate: cells produce more DHFR

eg. Glutamine synthetase: amplified by addition of methionine sulfoximine

• Non-dominant genes: target cells must have endogenous genes mutated or removed, only works with mutant cells. If target genes are successfully transfected with shuttle vector containing wild-type marker gene, this will allow cells to survive in specially treated media.

• Dominant genes: do not require mutant cells, can be used with any cell type. Dominant marker genes do not have to be mammalian, may be bacterial in origin, allows the cell to survive in special medium.

• Antibiotic resistance genes: shuttle vector contains antibiotic, allows for successful transfectants to survive.

• HGPRT- hypoxanthine guanine phosphoribosyl transferase

– Selection of cells in hypoxanthine, aminopterin and thymidine

– HAT media: contains hypoxanthine, aminopterin and thymidine

– HGPRT- cells can’t survive in HAT

Simple Precursors: purine, pyrimidine and

nucleotidesDNA

Hypoxanthine or

Guanine

Thymidine

TKHGPRT

Salvage pathway

Aminopterininhibition

De novo pathway

Genetic Markers…2. dominant genes – selection of any cell type, those

that acquire specific gene during transfection will survive

eg. Xanthine-Guanine Phosphoribosyl transferase (XGPRT) -present only in bacteria naturally-only transfected mammalian cells would be able to survive in media with xanthine, hypoxanthine mycophenolic acid

3. antibiotic resistance genes

• G418 is an aminoglycoside antibiotic, selects for expression of neomycin-resistant genes. G418 blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells.

• Blasticidin S is a peptidyl nucleoside antibiotic, selects for expression of the bsr gene. It specifically inhibits protein synthesis in both prokaryotes and eukaryotes through inhibition of peptide-bond formation in the ribosomal machinery.

2. Gene amplification

• involves increasing the number of copies of a specific gene per cell

→ increased synthesis of gene product

• can use inhibitors to select for cells with multiple copies of desired gene

• inhibitors do not induce gene amplification, but selects for cells that have undergone random gene amplification

•insert desired gene adjacent to dihydrofolate reductase or glutamine synthetase, up to 1000-fold increase in gene copy and corresponding protein expression

1. Transient gene expression• preliminary test of gene expression

• assayed from cell culture 1-3 days after uptake of DNA

Gene expression

cells are not genetically cells are not genetically stable and soon lose their stable and soon lose their expression ability expression ability

→ Ex. COS (monkey kidney cells) transfected with SV40 (2-5 x 105 copies of cloned genes/cell)

• SV40 DNA not replicated due to inactivation of origin of DNA replication, transfected DNA contains SV40 origin of replication

• leads to production of high copies of desired gene (2-5 x 105 copies of cloned genes/cell)

limited by frequency of DNA integrationlimited by frequency of DNA integration

ideal recombinant cells grow with ideal recombinant cells grow with doubling time of ~18 hours, is doubling time of ~18 hours, is genetically stable, and secretes a genetically stable, and secretes a recombinant protein at ~20% of total recombinant protein at ~20% of total cellular proteincellular protein

2. Stable gene expression

• DNA is integrated into the nucleus of the host cell, or efficient extrachromosomal replication system is maintained during cell division

Expression cassettes

• Expression signals required to ensure gene expression in mammalian cells

• Signals are common to wide variety of genes and used in a range of cell lines

• Expression cassette is a DNA sequence that contains signal sequences that are incorporated into a vector used to transfect mammalian cells

Signal sequences of an expression cassette include:

• a promoter to initiate transcription of the gene

→ want ‘strong promoters’ that allow for a high rate of transcription (SV40, adeno- or retroviral)

• ribosome-binding site for attachment of mRNA to ribosomes

• a transcription terminator sequence downstream of the expressed gene

Lecture 7 Animal Cell Biotechnology

Genetic engineering of animal cells

1. retroviral promoter and termination control regions

2. mammalian gene (globin cDNA)

3. selectable gene marker (neomycin resistance)

4. bacterial and mammalian origin of replication sitesButler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P106.