Applications of

Recombinant DNA Technology

© Sherry Fuller-Espie, 2002

I. Polymerase Chain Reaction
 A. Applications
 Polymerase Chain Reaction (PCR) has a wide
range of applications in many disciplines
– Molecular Biology/Research
– Diagnostics
– Genetic Counseling
– Criminology/Forensics
– Paternity testing
– Archeology
– Food testing
– Evolutionary studies
 B. Advantages over
traditional methodologies
 Fast and efficient amplification of specific DNA
sequences
 No requirement for cloning or subcloning
 Tiny amounts of material are usually sufficient
 Disease diagnoses will be greatly expedited by
PCR to identify microorganisms in infected people
who would prove falsely negative by other
diagnostic procedures
 Drawbacks:
– Can introduce errors in DNA during amplification
process
• Error-prone DNA polymerases
• New enzymes are reducing this tendency
– Vent polymerase vs. Taq polymerase
– Contaminants can give rise to false positives or
erroneous results
• Numerous controls must be included
• Controlled environment
 C. Procedure
 A specific DNA sequence is amplified using DNA
polymerase and oligonucleotide primers using many cycles
(25-30)
 ~106-fold amplification achieved
 Each cycle includes the following steps:
– Denaturation (92oC) (separation of dsDNA)
– Annealing (55oC) (primer binds to complementary regions)
– Extension (72oC) (primer is elongated by DNA polymerase)
 Exponential amplification of starting material achieved
 Required reagents for PCR:
– Template DNA (can be DNA or RNA)
• If RNA is used, an extra first step must be introduced
involving reverse transcription of RNA to produce DNA
template (= Reverse transcription PCR = RT-PCR)
– Two flanking oligonucleotide primers (excess)
– Nucleotides (excess)
– Heat-stable DNA polymerase
– Thermocycler to automate process (~6 minutes per
cycle, ~3 hours for 30 cycles)
 HIV integrates into T lymphocyte genome (CD4+
T cells)
– Integration can be detected using PCR
 Technique:
– Radioisotope tag is incorporated into amplified DNA
product of PCR (e.g. 3H-thymidine)
• DNA is separated from unincorporated nucleotides on glass
fiber disc
• Glass fiber disc is counted in a scintillation counter (beta
particle emission  photons generated in scintillant)
 Benefits:
– Earlier treatment can begin
– Especially useful for newborn determination of
HIV status
• Ab tests can’t rule out maternal IgG Abs
– Maternal IgG transfer transplacentally
– Can cause false positive result in newborn serum
• Have to wait several months to determine if Ab are
of fetal-origin
 #2: Lyme Disease – Detection of Borrelia
burgdorferi
– Caused by tick-borne spirochete
– Arthropod transmission
– Left untreated or not quickly enough:
• Arthritis and neurological complications (can be fatal)
– Problems:
• Very difficult to cultivate spirochetes in lab media
• Hard to see using conventional microscope
– PCR can detect as few as 5 spirochetes in a sample!!
• Can detect numerous strains (even if antigenic epitopes vary
 serological limitations)
 D. Examples

 #1: Detection of HIV in T-lymphocytes

– Serological techniques reguire humoral
immune responses to become activated for
successful detection of anti-HIV antibodies
(seroconversion)
• Acquired immune responses can take 10-14 days
before Ab titers reach maximum levels.
• Individual may test negative and transmit HIV
unknowingly (False negative)
 #3: Human Papilloma Virus (HPV)
– Causes genital warts and cervical cancer
– Tissue sample from cervix used in pCR
reaction
– Treatment can begin earlier
• Acyclovir
• Gancyclovir
 #4: Escherichia coli
toxins in food
– As few as 10 toxin-
producing E. coli cells can
be detected in a
population of 100,000
cells from soft cheese
samples
• Heat stable toxin (ST-1)
probes used
• Useful for detection and
control of food pathogens
– Other potential applications:
• Clostridium botulinum
• Campylobacter jejuni
• Vibrio cholerae
• Listeria monocytogenes
• Salmonella spp.
• Salmonella typhi
• Shigella spp.
• Giardia
• Trichinosis
 #5: E. coli in water
supplies
– Determination of water
quality
– Coliform count
• Filter water  Lyse
cells to release DNA 
PCR
• Primers specific for
lactose utilization genes
(DNAs from other
coliforms do not
respond to the primers)
 #6: Cystic fibrosis
– Autosomal recessive disease
– Defect in cystic fibrosis transmembrane conductance
regulator (CFTR) (chloride ion regulation)
– 70% of affected individuals have a 3bp deletion (∆508
– deleted phenylalanine from polypeptide)
• Detected by PCR using synthetic oligonucleotide primers to
amplify allele
– Then PCR product is denatured  Dot blot procedure 
probed with allele specific oligonucleotide (ASO)
– If ASO = WT CFTR
• Homozygous dominant  Dark spot (2 copies)
• Heterozygous  Light spot (1 copy)
• Homozygous recessive  No hybridization
– Can also use ∆508 ASO (Result = reversed)
– Screening
• To identify heterozygous carriers
• Parents can be informed of relative risks of producing
affected children
• Informed heterozygotes may have fetus screened
early in pregnency
II. Transgenic Animals
 A. Introduction
 Transgenic animals:

– Animals which have been genetically engineered to

contain one or more genes from an exogenous source.

– Transgenes are integrated into the genome.

– Transgenes can be transmitted through the germline to

progeny.

– First transgenic animal produced = “Founder Animal”

B. Introduction of foreign genes
into intact organisms
 Procedure is basically the same regardless of
which animal is involved.

 Integration usually occurs prior to DNA

replication in the fertilized oocyte.

– Majority of transgenic animals carry the gene in all of

their cells, including the germ cells. Transmission
to next generation requires germline integration.
– Some integration events occur subsequent to DNA
replication giving rise to mosaic animals which may
or may not contain the transgene in its germline.
 C. Procedure for Producing
Transgenic Mice

 Three different breeding pairs of mice are

required.
 First Breeding Pair:
– Fertile male + superovulated female
• Fertile male = stud (changed regularly to ensure performance)
• Superovulated female = immature female induced to
superovulate
– Pregnant mare’s serum (=FSH) on day 1
– Human Chorionic Gonadotropin (=LH) on day 3
• Mated on day 3
• Fertilized oocytes microinjected on day 4 with foreign DNA
construct.
• Microinjected oocytes are transferred to the oviducts of
surrogate mothers at end of day 4.
 Second breeding pair:
– Sterile male + surrogate mother
• Sterile male produced through vasectomy
• Surrogate mother must mate to be suitable recipient of
injected eggs
• Mated on day 3
• Microinjected oocytes from first breeding pair are
transferred to oviducts on day 4
• Embryos implant in uterine wall and are born 19 days later.
• Southern blotting techniques confirm presence and copy
number of transgenes.
 Third breeding pair:
– Foster parents
• Fertile male + female mated to give birth on same
day surrogate mother
• Serves as foster parent if caesarian section is
required for surrogate mother
D. Manipulation of Fertilized
Oocytes
See Slides
 E. Gene Expression in
Transgenic Mice
 In order to discriminate the products of the
injected gene from those of an endogenous
counterpart, the injected gene must be marked in
some way.
– Mini-genes where exons are deleted of cDNA where
introns are absent.
– Modification by insertion/deletion/mutagenesis of a
few nucleotides (e.g. the gain or loss of a restriction
endonuclease site).
– Hybrid genes where foreign epitopes are expressed on
transgenic products.
 F. Tissue-Specific Gene
Expression
 Generally, if a tissue-specific gene is expressed at all, then
it is expressed appropriately, despite the fact that it has
integrated at a different chromosomal location.
– Trans-acting proteins involved in establishing tissue-specific
expression are capable of finding their cognate sequences and
activation transcription at various chromosomal locations.

– Levels of expression vary between founder animals as

chromosomal position can influence accessibility of the transgenes
to these trans-acting transcription factors.

– Some founders do not express the transgene at all owing to

integration into heterochromatin domains where DNA is
methylated heavily (silent).
 G. Examples of Studies
Utilizing Transgenic Mice
 The Oncomouse
– c-myc oncogene + MMTV sequences  breast cancer
– Int-2 oncogene + viral promoter  prostate cancer

 To obtain abnormal expression of genes to study their

effects
– Rat growth hormone + cadmium-inducible metallothionein
promoter
– Transgenic mouse was much larger, but also suffered
complications with fertility and organ diseases
 To study developmentally regulated genes
 H. More Examples of Studies
Utilizing Transgenic Mice
 “Pharm” animals (transgenic livestock)

– Bioreactors whose cells have been engineered to

synthesize marketable proteins

– DNA constructs contain desired gene and appropriate

regulatory sequences (tissue-specific promoters)

– More economical than producing desired proteins in

cell culture
 I. Examples of Bioreactors
 Naked human Hb from  CfTCR in goats
pigs  Tissue plasminogen
 Human lactoferrin in activator (TPA) in goats
cows’ milk
 Human antithrombin III in
 Alpha-1-antitrypsin in
sheep goats
 Malaria antigens in goats
 HGH in mouse urine
(uroplakin promoters) (vaccine)
 Human antibodies in mice  Alpha-glucosidase in
(H and L chain tgenics  rabbits (Pompe’s disease
hybridomas)
 J. Transgenic Pigs for the
Production of Organs for
Transplantation
 Pig organs are rejected acutely due to the presence
of human antibodies to pig antigens.

 Once human antibodies are bound to pig organs,

human complement is activated and triggers the
complement cascade and organ destruction.

 Transgenic pigs with complement inhibitors have

been produced and are gaining feasibility as a
source of xenogeneic organs for transplantation.
III. The Knockout Mouse
 A. What is a Knockout
Mouse?

 A really good-looking mouse?

 A mouse in which a very specific

endogenous gene has been altered in such a
way that interferes with normal expression,
i.e. it has been knocked out.
 B. Why Produce KO Mice?
 To study effects of gene products,
biochemical pathways, alternative
(compensatory) pathways, and
developmental pathways

 To recreate human diseases in animals to

establish models to test the beneficial
effects of drugs or gene therapy.
 C. Procedure for Generating
a KO Mouse
 Gene alteration in KO mice is targeted to very specific
genes.

 DNA must integrate at precise positions in the genome.

 Integration of the altered gene takes place in embryonic

stem cells ex vivo.

 Verification of exact location of integration occurs before

the ESC is introduced into blastocysts to become part of
the developing embryo.
 D. Pluripotent ES Cells

 Pluripotent ES cells are undifferentiated early embryonic

cells derived from the inner cell mass of mouse
blastocysts.
 In vitro ES cells must be grown on a feeder layer of
fibroblasts to prevent them from differentiating.
 Introduction of the transgene is achieved by
electroporation of retroviral infection.
 The transgene must integrate via recombination, not
randomly.
 Cells transfected successfully can be identified prior to
injection into blastocysts.
 E. Specific Gene Targeting in ES
Cells
 Gene targeting can be achieved using gene
constructs designed for homologous
recombination. This technique can be used to
either:
– Knockout functional genes to study their contribution
to different developmental or disease processes (null
mutations)
• Genes encoding β2m, MHC class I and II. CD2, Ii, TCR, Ig,
IL-4, IL-2, FcεR, TAP1/2, RAG-2,and many more (>100)!
– Replace a functional gene for a mutated/non-functional
gene to restore wild type phenotype .
• Gene encoding HGPRT in mice deficient for HGPRT (called
Lesch-Nhyan syndrome in humans).
 F. DNA Constructs for
Recombination
 DNA vectors contain the gene of interest which
has been interrupted with an antibiotic resistance
gene (hygromycin resistance, or G418 resistance).

 To ensure targeted integration has occurred, the

flanking DNA contains the thymidine kinase gene.
If TK integrates (random insertion), then the
transfected cells die when grown in selective
media (gancyclovir).
 G. Selection of Targeted ES
Cells
 Gancyclovir resistant and G418 resistant ES cells
grow into small clumps on top of feeder cells.
 The colonies of cells can be “picked” off and
transferred to new wells (at 0.3 cells per well
seeding density) containing feeder cells.
 When sufficient numbers of cells are obtained,
they are:
– Frozen for safe storage
– Analyzed by Southern blotting or PCR to determine
nature of integration event
– Microinjected into the blastocoel cavity of blastocysts.