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.

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).

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 pigs

Human lactoferrin in cows’ milk

Alpha-1-antitrypsin in sheep

HGH in mouse urine (uroplakin promoters)

Human antibodies in mice (H and L chain tgenics hybridomas)

CfTCR in goats Tissue plasminogen

activator (TPA) in goats Human antithrombin III in

goats Malaria antigens in goats

(vaccine) Alpha-glucosidase in

rabbits (Pompe’s disease

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 2m, MHC class I and II. CD2, Ii, TCR, Ig, IL-4, IL-2, FcR, 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.

III. Dolly and the Advancement of Animal Cloning

How Was Dolly Made?