BT620 (2019-2020, Semester II)

In this section (post mid-semester examination), we will learn

about recombinant DNA engineering using animal cells.

Some of the techniques used to engineer cells will be familiar

to you while studying about recombinant DNA engineering
tools using plants/plant cells. Some will be new. The
applications will be new.

The major aim here is:

(i) to produce ‘authentic’ human proteins in huge quantities,
mostly for therapeutic purpose,
(ii) to create human-relevant animal models of diseases.
I have followed two books:
(i) Principles of Gene Manipulation and Genomics (S. B.
Primrose and R. M. Twyman), 7th edition, Blackwell
Publishing, 2006; and
(ii) Molecular Biotechnology. Principles and Applications of
Recombinant DNA (B. R. Glick, J. J. Pasternak and C. L.
Patten), 4th edition, ASM Press, 2009.

In addition, some relevant examples from recent research

papers have been used to illustrate concepts. References to
these papers will be cited on the relevant slides.

Some explanation, mostly from the books listed above, will

follow the slides.
Why use animal cells?
• Authentic post-translational modifications

How different from plants cells?

• Most animal cells are restricted in terms of
their developmental potential

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Background:
Mammalian cells are the most widely used hosts for gene delivery, since
they allow the production of recombinant human proteins with authentic
post-translational modifications that are not carried out by bacteria, yeast, or
plants. Indeed, mammalian cells are cultured on a commercial scale for the
synthesis of many valuable products, including antibodies, hormones,
growth factors, cytokines, and vaccines.

For now, we will talk of methods for the introduction of DNA into somatic
cells, i.e. cells that do not contribute to the animal germline.
Unlike in plants, animal somatic cells are restricted in their developmental
potential. Mostly, any part of the plant can be transformed with a foreign
gene and made to give rise to a transgenic plant where each cell of the plant
contains the foreign gene. This is not possible with animal cells unless
special techniques are used. We will talk of techniques to generate
transgenic animals later.

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Gene transfer strategies
• Result in transformation: Change of recipient
cell’s genotype, caused by the acquired DNA,
the transgene

1. Direct DNA transfer

2. Transfection
3. Transduction
4. Bactofection

2
Background:
Gene transfer to animal cells can be achieved using four broad types of
delivery mechanism. Two of these are described as biological mechanisms
because the target cells need to be infected with a biological delivery vector,
such as a virus or bacterium, which carries the exogenous genetic material.
Delivery using a viral vector is known as transduction, and many different
viruses have been adapted as gene-delivery vectors. The transgene is
therefore delivered as part of a recombinant viral genome, exploiting the
virus’s natural ability to infect and transfer nucleic acid into animal cells.
We will look at viral vectors in detail later.

Delivery using bacterial vectors relies on bacteria which also invade animal
cells. Bacterial gene delivery is sometimes termed bactofection.

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1. Direct DNA transfer
a. Microinjection
b. Particle bombardment
c. Electroporation
d. Ultrasound

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2. Transfection
a. Chemical
DNA-calcium phosphate coprecipitation method
1. Fresh precipitation
2. Suitable for cells growing in monolayers
3. Not suitable for stable transfection
Alternatives
1. DEAE-dextran
2. Polylysine
3. Polyamines, PEI and dendrimers
4. Stimulus-sensitive polymers 4
Lipofection
Cells + DNA + Lipofection agent

Internalization by endocytosis

Transported to nucleus

Expression

• Low toxicity method

• High transfection efficiency
• Suitable for stable and transient transfections
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Lipofection

2-4 ml Transfectin 0.2-1 mg plasmid

per 50 ml of serum- DNA per 50 ml of
free medium serum-free medium

Mix equal volumes

of Transfectin and
DNA solutions

Incubate for 20 min to

form DNA-liposome
complexes
Plated cells
Mix, add DNA-liposome
complexes directly to cells
100 ml/24-well plate

Aspirate media
from cells
Incubate overnight
and assay
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Background:
The other two delivery mechanisms are described as non-biological
because biological delivery vectors are not required.
To distinguish such methods from those involving infection with a bacterium
or virus, the term transfection is used.
In chemical transfection methods, cells take up DNA from their
surroundings when the DNA is presented as a synthetic complex – either a
complex with an overall positive charge, allowing it to interact with the
negatively charged cell membrane and promote uptake by endocytosis; or a
lipophilic complex which fuses with the membrane and deposits the
transgene directly into the cytoplasm. The latter process is known as
lipofection and is one of the most common methods of transfection. The
use of serum-free medium ensures the absence of DNases so that the
foreign DNA is not degraded before it enters the cell.

In physical transfection methods, naked DNA is introduced directly into the

cell by exploiting a physical force. These are listed under Direct DNA
transfer methods and you have learnt about them earlier.

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c. Delivery vehicles
Phospholipids
Haemoglobin-free erythrocytes
YAC transgenic cells

• DNA is packaged inside the delivery vehicle which

interacts with the cell membrane and is internalized
• Gentle procedure
• Difficulty in encapsulating DNA
• Poor transfection efficiency
Liposomes
Cationic/neutral lipid mixtures
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 TRANSFECTION INTEGRATION
DNA Cell Incorporation
into genome
Stable Transient

Transient transfection
•Properties of the cells are transiently altered
•Transgene is maintained in an extrachomosomal
state in the nucleus
•Transgene lacks an origin of replication that
functions in the host cell
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Stable transfection
•Transgene is integrated into the host cell genome
•The new genetic locus is inherited by all clonal
descendants – Creation of cell line
Uses
Transient transfection
1. Short-term effects
Stable transfection
1. Long-term analytical experiments
2. Production of large quantities of recombinant
proteins 9
Background:
The transformation of animal cells occurs in two stages, the first involving the
introduction of DNA into the cell (the transfection stage), and the second involving its
incorporation into the nucleus, often by integration into the host chromosome.
Transfection is much more efficient than integration, hence a large proportion of
transfected cells never integrate the foreign DNA they contain.

The DNA is maintained in the nucleus in an extrachromosomal state. If it does not

contain an origin of replication that functions in the host cell, it persists for just a short
time before it is diluted and degraded. This is known as transient transformation/
transfection, which means that the properties of the cell are changed by the introduced
transgene, but only for a short duration. A majority of experiments are carried out using
transient transfection.

In a small proportion of transfected cells, the DNA will integrate into the genome,
forming a new genetic locus that will be inherited by all clonal descendants. This is
known as stable transformation, and results in the formation of a “cell line” carrying
and expressing the transgene. Since integration occurs only in a small number of cells,
these rare, stably transformed cells must be isolated from the large background of
non-transformed and transiently transformed cells by selection.

Next, we will look at the methods to select these transformed cells.

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Selection of transformants
1. Availability of endogenous markers
Production of nucleotides in mammals

X
De novo pathway
[Blocked by aminopterin]
Salvage pathway

Cells with functional Hprt

and Tk genes can survive

Select viable cells

with HAT medium
[Hypoxanthine, aminopterin, thymidine]

Transgene confers a detectable phenotype

on the selected cells
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Background:
In mammals, nucleotides are produced via two alternative routes, the de
novo and salvage pathways (as shown in the following slide). In the de
novo pathway, nucleotides are synthesized from basic precursors such as
sugars and amino acids, while the salvage pathway recycles nucleotides
from DNA and RNA. If the de novo pathway is blocked, nucleotide synthesis
becomes dependent on the salvage (redundant) pathway, and this can be
exploited for the selection of cells carrying functional Hprt and Tk genes.

The drug aminopterin blocks the de novo synthesis of both inosine

monophosphate (IMP) and thymidine monophosphate (TMP) by inhibiting
key enzymes in the de novo pathway. Cells exposed to aminopterin can
thus survive only if they have functional Hprt and Tk genes and a source of
hypoxanthine and thymidine. Hprt+ and Tk+ transformants can therefore
both be selected using HAT medium, which contains hypoxanthine,
aminopterin, and thymidine.
You would have come across HAT medium while studying about hybridoma
technology.

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 HPRT: hypoxanthine-guanine phosphoribosyltransferase
DHFR: Dihydrofolate reductase

TK: Thymidine kinase

Note: There is no salvage pathway for CTP 10-2

1. Availability of endogenous markers
•Confers a property that is already present in wild-type cells
•Only works in mutants with non-functional host gene
•Markers are present in redundant metabolic pathways

Marker Product
Ada Adenosine deaminase
Aprt Adenosine phosphoribosyl
transferase
Cad Multifunctional enzyme
Hprt Hypoxanthine-guanine
phosphoribosyl transferase
Tk Thymidine kinase
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Background:
The purpose of using endogenous markers is to be able to select those
cells which have successfully taken up the transgene (foreign gene) and
given rise to transformants (transformed cells). Since the transgene may not
have any detectable phenotype (what this means is that if the transgene
codes for an enzyme, you can assay its activity and know if it has been taken
up by the cell or not. But if it codes for structural proteins like collagen, etc.,
you cannot measure their ‘activity’, i.e. you will never know if your cell has
picked up the transgene or not), a good idea to look for correctly transformed
cells is by fusing the gene of your interest with a marker gene, e.g. Hprt or
Tk.

However, all cells have functional Hprt and Tk (wild type cells), so whether
or not the cell has picked up your transgene, the cell will survive in HAT
medium (see background material of the previous slide).

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Background:

 So what you need to start with are mutant cells, i.e. which lack Hprt and
Tk (Hprt-, Tk-). If you block the de novo pathway in these cells by adding
aminopterin in the medium (the “A” of HAT medium), the only way these cells
can survive is if they have functional Hprt and Tk, i.e. the salvage pathway is
activated.

And how is the salvage (redundant) pathway activated?  By providing the

genes for Hprt and Tk along with the gene of your interest (transgene). If and
when the cell picks them up and you expose these cells to HAT medium
(which also contains hypoxanthine and thymidine, required for the enzymatic
synthesis of IMP and TMP by Hprt and TK, respectively), the only cells which
will survive are cells which have picked up the transgene!

We will look at other methods of selection of transformants in the next class.

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