19.02.

2025

GENETIC
ENGINEERING
Molecular Biology
Lecture 4

© 2024 Aleksandra Śliwa PUMS

What is genetic engineering?

Initially referred to various

techniques used for the
modification or manipulation of
organisms through the processes of
heredity and reproduction.

As such, the term embraced both

artificial selection and all the
interventions of biomedical
techniques, among them
-artificial insemination,
-in vitro fertilization,
-cloning, and
-gene manipulation.

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Example of inbred mice: BALB

The strain obtained by Halsey J. Bagg of Memorial
Hospital, New York.
• BALB/c substrains are "particularly well known for the production
of plasmacytomas on injection with mineral oil," an important
process for the production of monoclonal antibodies.

• Most substrains have a "long reproductive life-span", are noted for

displaying high levels of anxiety and for being relatively resistant
to diet-induced atherosclerosis, making them a useful model for
cardiovascular research.

Example of inbred mice: nude

The nude mouse (or athymic nude mouse) was
obtained in 1966 by S. P. Flanagan at the Institute
of Animal Genetics in Edinburgh.
The nude mice arose spontaneously in an albino strain of mice.

Homozygous nude mice (nu/nu) were characterized by their macroscopic morphology,

specifically the fact that they failed to develop fur after birth. Flanagan also pointed out
that the nude mice exhibited decreased reproductivity, increased postnatal mortality, and
the fact that virtually all of the mice developed systemic toxoplasmosis that he attributed
to a possible „inborn error in metabolism”.

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Example of inbred mice: nude

• The nude mouse is homozygous for the mutant form of the
Foxn1 gene (nu/nu) – an evolutionarily conserved winged-
helix transcription factor (Foxn1), that is critical for
controlling thymus development and for regulating the
expression of specific hair keratin genes.

• It is known that the original nude mouse phenotype (Foxn1nu) is

caused by a single base pair deletion in exon 3, which leads to
a frameshift and a premature stop codon (lack of DNA binding
domain).

Example of inbred mice: nude

Vacanti Mouse

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Example of inbred mice: nude

What is genetic engineering?

In the latter part of the 20th century, the term came to refer
more specifically to methods of recombinant DNA technology

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What is genetic engineering?

Genetic engineering is the artificial manipulation,

modification, and recombination of
DNA or other nucleic acid molecules
in order to modify an organism or population of organisms.

Recombinant DNA

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Recombinant DNA
Expression vector

The plasmid performs different functions in

prokaryotic versus eukaryotic cells. The
portions of the plasmid denoted in yellow
represent sequences relevant to bacteria:
PSV40e is a promoter from simian virus 40
that will dictate the transcriptional start site
for the bacterial gene Kanr.

- Kanr is a bacterial gene encoding a protein

that will confer antibiotic resistance to
kanamycin.
- HSV TK poly A encodes a signal (obtained
from the herpes simplex virus gene for
thymidine kinase) that will cause a poly(A)
tail to be put onto the 3′ end of the
bacterial gene during transcription.
- pUC is a DNA sequence that serves as a
starting point, or origin, for bacterial
replication.

An Introduction to Biotechnology
The Science, Technology and Medical Applications
2014, Pages 237–274

Recombinant DNA
Expression vector

The portions shown in green

represent sequences relevant to
eukaryotic – mammalian cells:
- PCMV IE is a promoter for
“immediate early” genes in the
cytomegalovirus genome. This is
a strong promoter in mammalian
cells, meaning it supports a large
amount of transcription initiation.
- EGFP encodes the gene for an
enhanced green fluorescent
protein.
- SV40 poly A encodes a signal for
polyadenylation of mRNA
transcripts.

An Introduction to Biotechnology
The Science, Technology and Medical Applications
2014, Pages 237–274

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Recombinant DNA
Gene Construct = insert
• In adding a restriction site to an
amplicon, the PCR steps are the same
as in regular PCR. However, extra bases
have been added to the 5′ end of each
primer. These bases will not pair with
the genomic DNA.

• The first cycle of PCR will yield dsDNA

fragments with the added cut site and
spacer still unpaired.
• The second cycle of PCR will yield a
completely paired dsDNA fragment on
the side where the primer from the
previous cycle is located.
• The third cycle of PCR will yield the first
completely paired dsDNA fragments.

An Introduction to Biotechnology
The Science, Technology and Medical Applications
2014, Pages 237–274

Recombinant DNA

How to put the gene constuct and the vector together?

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Recombinant DNA
Restriction enzymes

source:http://tle.westone.wa.gov.au

Recombinant DNA
Restriction enzymes
Endonuclease – nuclease that
cleaves phosphodiester bonds within a
nucleic acid chain.

These enzymes are found

in bacteria and archaea and provide a
defense mechanism against
invading viruses.
Inside a prokaryote RE selectively cut
foreign DNA and host DNA is protected
thanks to a methyltransferase
that modifies the prokaryotic DNA.
Together, these two processes form
the restriction modification
system.

The targets of a phosphatase and a

nuclease

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Recombinant DNA
Restriction enzymes
• Restriction enzymes are traditionally classified into four types on the
basis of subunit composition, cleavage position, sequence specificity and
cofactor requirements.
• However, amino acid sequencing has uncovered extraordinary variety
among restriction enzymes and revealed that at the molecular level,
there are many more than four different types.

www.neb.com

Recombinant DNA
Restriction enzymes
• Type II enzymes cut DNA at defined positions close to or within
their recognition sequences.

• They produce discrete restriction fragments and distinct gel

banding patterns, and they are the only class used in the
laboratory for routine DNA analysis and gene cloning.

• Type II enzymes are a collection of unrelated proteins of many

different sorts.

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Bacterial
plasmid

Restriction site
5′ 3′
G A AT T C
DNA C T TA A G
3′ 5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
3′ 5′
5′ 3′

5′ 3′
3′ 5′
Sticky end

3′ 5′
5′ 3′

5′ 3′
3′ 5′
Sticky end
5′
3′
2 Base pairing of sticky
ends produces various 3′
5′
combinations.
Fragment from different
DNA molecule cut by the
same restriction enzyme
5′ 3′ 5′ 3′ 5′ 3′
G AATT C G AATT C
C T TAA G C T TAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination

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Figure 19.6c 5′ 3′ 5′ 3′ 5′ 3′
G AATT C G AATT C
C TTAA G C T TAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination
3 DNA ligase
seals the strands
5′ 3′

3′ Recombinant DNA molecule 5′

Recombinant
plasmid

Bacterial
plasmid

Restriction site
5′ 3′
G AAT T C
DNA
C T T AAG
3′ 5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
3′ 5′
5′ 3′

5′ 3′
3′ 5′
Sticky end
5′
3′
2 Base pairing of sticky
ends produces various 3′
combinations. 5′
Fragment from different DNA molecule
cut by the same restriction enzyme
5′ 3′ 5′ 3′ 5′ 3′
G AAT T C G AAT T C
C T TA A G C T TAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination
3 DNA ligase
seals the strands.
5′ 3′

3′ Recombinant DNA molecule 5′

Recombinant
plasmid

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Recombinant DNA

http://cloninginfo101.weebly.com/

Plasmid or Viral Vectors?

Both!

Addgene.com

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What is genetic engineering?

Genetic engineering is the artificial manipulation,
modification, and recombination of
DNA or other nucleic acid molecules
in order to modify an organism or population of organisms.

What would a genetically modified cell look like?

Genetically modified cells

Host cel types used for production of biopharmaceuticals

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Genetically modified cells

Bacterial cells used for protein expression

Genetically modified cells

E.coli used for protein expression
Most common microbial species to produce heterologous proteins
of therapeutic interest
Heterologous protein: protein that does not occur in host cells

Major advantages of E. coli

- Served as the model system for prokaryotic genetics
- Its molecular biology is well characterized
- High level expression of heterologous proteins:
- High expression promoters (~30 % of total cellular protein)
- Easy and simple process: rapid growth, simple and inexpensive media,
appropriate fermentation technology, large scale cultivation
Major disadvantage of E. coli
- Glycosylation pattern usually differs from the pattern observed in the
native glycoprotein

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Genetically modified cells

Eukaryotic cells used for protein expression
Major advantage: Suitable for production of glycoproteins

Examples: Chinese Hamster Ovary (CHO) and Baby Hamster Kidney (BHK)

Major disvantages:
- Very complex nutritional requirements: growth factors
- Slow growth rate: long cultivation time
- Far more susceptible to physical damage
- Increased production cost
- Complicated purification procedure

Typical proteins produced in animal cells:

EPO, tissue plasminogen activator (tPA),
Interferons, Immunoglobulins, Blood
factors etc

Genetically modified cells

Insulin production in E.coli and S.cerevisiae
• Previously isolated from swine’s pancreas – the
method was expensive and insuline had several side
effects.

• The first therapeutic protein produced by E. coli:

Human insulin (Humulin) in 1982.

The first drop of biosynthetic insulin,made by Eli

Lilly & Co. using recombinant DNA technology.

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Genetically Modified Animals

Cells as Bioreactors
Bacteria Yeast Fungi Plants Cell Animals
cultures
Speed ++++++ ++++ +++++ ++ +++ +

Total costs ++++ ++ +++ ++++++ + +++++

Post- + ++ +++ ++++ ++++++ +++++

translational
modifications

Up-scaling ++++ ++ +++ ++++++ + +++++

Regulations +++++ ++++ +++ ++ ++++++ +

Słomski, Beijing 2010

Some recombinant proteins approved for human use

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Genetically Modified Animal Models

To create embryos in which the genome is permanently altered and the
transfected DNA is permanently and irreversibly incorporated into the
genetic material of the host cells, there are at least three important steps to
be surmounted:
-Produce a gene construct in vitro that is functional in accordance with the hypothesis
of the proposed study.
-Cause the required DNA to cross the plasma membrane, the cytoplasm, and reach
the nucleus.
-Cause the exogenous DNA to be integrated into the genome of the host cell and
to be passed on unaltered to the following generations.

Animal Models for the Study of Human Disease

2013, Pages 811–831; www.pdn.cam.ac.uk

Genetically Modified Animal Models

Gene Construct
• The construct can be designed to be inserted randomly into the
genome of the animal, which is called transgenesis by
addition, or can be designed to be inserted into the genome at a
specific targeted site, into the correct position of a determined
chromosome, which is called transgenesis by homologous
recombination.

• Gene construct elements:

-promoter
-a site of transcription initiation
-a site of polyadenylation and
-a site of transcription termination

Animal Models for the Study of Human

Disease 2013, Pages 811–831

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Genetically Modified Animal Models

Insertion of the DNA of Interest into Cells
Microinjection is the most widely used
method in transgenesis for gene addition.
DNA in solution is physically injected into
the cell nucleus.
• 100% efficiency in carrying the DNA
into the nucleus of somatic cells.
• Widely used for the insertion of gene
vectors into fertilized oocytes, and it has
a success rate of 4–8% of animals
born with the transgene integrated
into the genome.

Animal Models for the Study of Human

Disease
2013, Pages 811–831

Genetically Modified Animal Models

Integration
• Integration of exogenic DNA into the
host genome occurs with an extremely
low efficiency.

• It is believed that the integration of

exogenic DNA occurs as a
consequence of errors made in repair
of breaks dsDNA.

• Integration of transfected DNA typically

occurs in random locations in the
genome (may disrupt, prevent, or
impede).

Animal Models for the Study of Human Disease

2013, Pages 811–831

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Types of Genentically Modified Animals

1. Transgenic animals
2. Knockout animals
3. Conditional knockout animals
4. Knockin animals

Genetically Modified Animals

Transgenic models have diverse functions in research, in which the inserted gene may
have its effects evaluated in vivo during the developmental stages, as in the
evolution of diseases, in studies of mutations, in the search for therapies etc.

Industrially, the transgenic model can be used to obtain commercial productivity

characteristics, and disease resistance, and can be used as a bioreactor in the
production of biopharmaceuticals.

Animal Models for the Study of Human

Disease
2013, Pages 811–831

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Genetically Modified Animals

Transgenic Animals
Transgenic animals are those in which an exogenous gene was
artificially inserted and stably incorporated into the genome
of every cell of the organism and that can be transmitted to their
descendants.

The first transgenic mouse had the genome sequence of the

Maloney leukemia virus inserted into its genome (Jaenisch R,
PNAS 1976).

The animal generated from this egg cell, known as a transgenic founder, is heterozygous for the transgene, i.e.
one of the chromosomes has incorporated the transgenic DNA, but not the homologous chromosome.
Consequently, only half of the animals obtained from the F1 mating will also be heterozygous.

+
Animal Models for the Study of Human
Disease
2013, Pages 811–831

Genetically Modified Animals

Transgenic Animals - Bioreactors
In order for the transgenic animal system
to be practically useful, the target protein
must be easily and simply separable
from the animal without any injury.

• E.g. to produce a target protein in a

mammary gland - the protein of
interest is linked to milk-specific
regulatory elements to generate the
transgene).
• Easy recovery of a target protein from
milk

[email protected]

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ATryn – first drug produced by transgenic animal

approved by FDA and EMEA (2009, 2006)

One goat may replace

90,000 of a blood donors.

Genetically Modified Animals

Transgenic Animals - Bioreactors

Słomski, Beijing 2010

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Genetically Modified Animals

Transgenic Animals - Bioreactors
Bacteria Yeast Fungi Plants Cell Animals
cultures
Speed ++++++ ++++ +++++ ++ +++ +

Total costs ++++ ++ +++ ++++++ + +++++

Post- + ++ +++ ++++ ++++++ +++++

translational
modifications

Up-scaling ++++ ++ +++ ++++++ + +++++

Regulations +++++ ++++ +++ ++ ++++++ +

Słomski, Beijing 2010

Genetically Modified Animals

1. Transgenic Animals

Silkworm (Bombyx mori) -

natural silk manufacturer for
textile industry.
• Cocoon – potential source of high
amounts of recombinant proteins
• Short time of generation
• Production of vaccines - cholera
toxin B subunit fusion protein linked
with human insulin B chain peptide
at levels up to 0.97 g/l of
hemolymph

Słomski, Beijing 2010

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Genetically Modified Animals

Types of polypeptides produced in

transgenic animals
Rabbit Pigs
•human IGF-1, protein C,
•human tissue plasminogen activator, human factor VIII
•erythropoietin, Sheep
•α-glucosidase, human factor VIII, Cows
•factor NGF-β, human factor IX, human lactoferrin,
•protein C, human α-1- antitrypsin, human erythropoietin,
•human growth hormone, fibrinogen human serum albumin
•rotavirus inner core proteins,
•human factor VIII, Goat
•human alpha 1,3 fucosyltransferase •human lactoferrin,
•E2–CSFV vaccine,
•human tissue plasminogen
activator,
•human antithrombin III,
•human monoclonal antibodies,
•growth hormone

Słomski, Beijing 2010

Genetically Modified Animals

2. Knockout Animals
The great contribution of these
models was in the study of gene
function through the phenotype
presented due to the effect of allelic
deletion.
• Unlike the transgenic model
produced by the addition of
random exogenous DNA, the
knockout model is obtained by
targeted insertion.
• This targeted integration is
performed through the
mechanism of homologous
recombination and has an
extremely low success rate.

Animal Models for the Study of Human Disease

2013, Pages 811–831

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Genetically Modified Animals

3. Conditional Knockout Animals

• Deletion of genes essential for

development can result in
embryonic lethality.
• One possible strategy to
circumvent this problem is the use
of the conditional deletion system,
which involves gene deletion under
specific conditions.
• The main strategy is known as the
Cre–Lox system, which enables
to generate tissue-specific and
inducible knockouts and thereby
have exquisite control over the
location and timing of gene
expression.

Animal Models for the Study of Human Disease

2013, Pages 811–831; www.jax.org

Genetically Modified Animals

4. Knockin Animals
The same techniques are used to introduce new genetic
elements into specific sites, known as knockin.

One application of this technique is to study the expression of

molecules, identifying cells that express and modulate expression,
using reporter molecules for this (β-galactosidase, GFP).

Since the discovery of GFP, derivatives that fluoresce in different

colors have been engineered.
Expression of a lacZ gene can be followed in the mouse by
staining for b-galactosidase.

Animal Models for the Study of Human Disease

2013, Pages 811–831; Photo: Robb Krumlauf, Stowers
Institute for Medical Research

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Genome editing
• Genome editing can be used to modify any gene sequence of
interest in either cells or whole organisms.

neb.com

Genome editing - meganuclease

• First genome-editing strategy was based on using I-SceI yeast
meganuclease, a rare-cutter endonuclease with a recognition site
of 18 base pairs responsible for intron homing in yeast
mitochondria.

PLoS Biol. 2014 Jun

10;12(6):e1001877

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Genome editing - meganuclease

Mol Cell Biol. 1995 Apr; 15(4): 1968–1973

It was demonstrated that double-strand breaks can be initiated by

the I-SceI endonuclease at a predetermined location in the mouse
genome and that the breaks can be repaired with a donor molecule
homologous regions flanking the breaks.

Genome editing – zinc finger nuclease

Zinc Finger Technology
In a pioneering study, Guerts, Jacob and Buelow, and colleagues
used zinc-finger nucleases (ZFN) to produce the world´s first
knockout rats.

Geurts et al. demonstrated that a single injection of DNA or mRNA

encoding ZFNs into the one-cell rat embryo leads to a high
frequency of animals carrying 25–100% disruption at the target
locus.

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Genome editing
Zinc Finger Technology
In a pioneering study, Guerts, Jacob and Buelow, and colleagues
used zinc-finger nucleases (ZFN) to produce the world´s first
knockout rats.

The methodological approach was based on the use of a DNA

endonuclease domain from the bacterial restriction enzyme FokI,
engineered with zincfinger domains with known DNA-binding
capacity, which was used to target and cleave a specific genome
location.

Genome editing - ZNF

Zinc Finger Technology
Zinc finger nuclease (ZFN) technology utilizes a FokI nuclease as
the DNA-cleavage domain and binds DNA by engineered
Cys2His2 zinc fingers. Specific zinc fingers recognize different
nucleotide triplets and dimerize the FokI nuclease. The activated
nuclease introduces a double stranded break between the two
distinct zinc finger binding sites, which prompts recombination
and modification of the genome.

addgene.com

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Genome editing - TALEN

TALEN Technology

TALEN is a second-generation artificial Transcription Activator-like

Effector Nuclease

TALEs were first

discovered in bacteria
Xanthomonas oryzae.

bacterial blight bacterial leaf streak

knowledgebank.irri.org

Genome editing - TALEN

TALEN Technology
Transcription activator-like effector nuclease (TALEN) systems are a
fusion of TALEs derived from the Xanthomonas spp. to a
restriction endonuclease FokI. By modifying the amino acid
repeats in the TALEs, users can customize TALEN systems to
specifically bind target DNA and induce cleavage by the nuclease
between the two distinct TAL array binding sites.

addgene.com

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Genome editing – CRISPR

CRISPR Technology
RNA-guided endonucleases (RGEN) utilize a short guide RNA
(gRNA) to recognize DNA, bind an endonuclease, and induce site
specific cleavage.
CRISPR genome editing systems allow users to design gRNA which
target their DNA sequence of interest.

addgene.com

Gene editing - CRISPR/Cas9

Clustered Regularly Interspaced Short Palindromic Repeat

3 distinct types of bacterial CRISPR systems

identified so far:
Type I, II, III
Type II is the basis for current genome engineering
applications
From Streptococcus pyogenes
The S. pyogenes system is orthogonal to the native E. coli system

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CRISPR/Cas9 – legal battle

Feng Zhang from the

Broad Institute of MIT & Harvard
Jennifer Doudna from
University of California, Berkeley

The future of genome engineering…

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THE END

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