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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 toxinproducing 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    

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 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.