Genetic Technology & Genetic Therapy

What is a Clone?

• A clone is an identical copy of a DNA segment, a whole cell, or a complete organism, all derived from a single ancestor.

• Cloning refers to the process of producing identical copies of molecules, cells, or organisms.

How to Create a Clone

• Plants can be cloned from single cells.

• In the 1950s, Charles Steward grew individual carrot cells in the lab.

• Cells grew into a ball of undifferentiated cells called a callus.

• In different media, calluses grew into normal carrots.

Plant Cloning

• Plants can be cloned from various tissues including leaves, roots, and stems.

• All plant cloning involves:

  • De-differentiation: Forgetting what type of cell they are.

  • Re-differentiation: Specializing using different media.

Animal Cloning

• Animal cloning is more complex than plant cloning.

• Traditional cloning involved mating two organisms with desirable traits to produce offspring.

• The offspring with the best combination of traits were then used as parents.

• This has been happening in agriculture for centuries through artificial selection.

Examples of Historical Animal Cloning

• Breeds such as European Age Breeds, Modern Wolves, Herding, Hound, Sporting, Working, and Ancient Asiatics are examples of historical animal cloning.

Newer Cloning Methods: Embryo Splitting

• This is a variation on the natural occurrence of twins.

• First done in 1902 by Hans Spemann.

• It’s an artificial “twinning” process.

• Procedure:

  • Collect an egg from a female and fertilize it in vitro.

  • Allow the embryo to develop into 4-8 cells.

  • Separate all cells into 4-8 embryos.

  • Implant into surrogates.

  • All offspring are clones.

Newer Cloning Methods: Nuclear Transfer

• This method is more technically difficult but yields a larger number of cloned offspring.

• The nucleus from an egg cell is removed.

• The nucleus from another cell is inserted.

• First accomplished in 1952 by Robert Briggs and Thomas King on the Northern Leopard Frog.

Early Nuclear Transfer

• Donor egg: nucleus removed.

• Embryo cell: nucleus removed.

• Embryo nucleus implanted into donor egg.

• Normal growth and development occur.

Nuclear Transfer Pioneers

• 1958 – John Gurdon used intestinal cells to create a clone.

• Mammalian eggs are smaller and more difficult to work with.

• 1975 – J. Derek Bromhall created first mammalian embryo by nuclear transfer.

Nuclear Transfer – Dolly the Sheep

• 1997 - First nuclear transfer using adult DNA (Somatic Cell Nuclear Transfer).

• Nuclear fusion was used rather than injection.

• Low success rate:

  • 277 fusions

  • 29 embryos

  • 13 surrogates

  • 1 full-term pregnancy

The Process of Cloning Dolly the Sheep

• A donor cell is taken from a sheep's udder.

• An egg cell is taken from an adult female sheep, and its nucleus is removed.

• The donor nucleus is fused with the enucleated egg cell using an electric shock.

• The fused cell begins dividing normally.

• The embryo develops normally into a lamb (Dolly!).

• The embryo is placed in the uterus of a foster mother.

What Happened to Dolly?

• She lived a pampered life and had lambs of her own.

• Euthanized at 6½ years of age due to arthritis, lung tumor, and early aging.

• This was possibly due to telomere shortening or cell differentiation.

Nuclear Transfer These Days

• Egg cell taken from donor, nucleus removed.

• Adult cell taken from animal to be cloned.

• Nucleus from adult cell injected into empty egg.

• Egg ”reprograms” the nuclear material.

• Encouraged to divide by electric shock.

• Implanted into surrogate.

• Much higher success rate than nuclear fusion.

• Routinely used in agriculture for sheep, cows, goats, and pigs.

Cloning Genes

• Does not involve cloning whole organisms.

• Involves Recombinant DNA Technology.

• Produces clones of DNA molecules.

• Involves transfer of genes between species.

• Used to find genes and map them.

• Identifies carriers of genetic disorders.

• May be used in gene or genetic therapy.

DNA Cloning Requirements

• A way to cut the DNA consistently.

• A carrier molecule to transfer the DNA into your cell of choice.

• A host cell into which to transfer the DNA.

Restriction Enzyme DNA Cutting

• Bacterial natural defense enzymes.

• Hunt out specific sequences in DNA and cut both strands.

• Usually 4-8bp recognition sequences.

• Some enzymes make a blunt cut, while others create a sticky end.

• Prevents infection of foreign DNA.

• Hundreds of Restriction Enzymes (REs).

• Each has its own specific recognition sequence.

Restriction Enzyme Cutting Patterns

• Examples:

  • EcoRI (Source: Escherichia coli)

  • HindIII (Source: Haemophilus influenzae)

  • BamHI (Source: Bacillus amyloliquefaciens)

  • Sau3A (Source: Staphylococcus aureus)

  • HaeIII (Source: Haemophilus aegypticus)

• Enzymes can produce sticky ends or blunt ends.

Sticky Ends in Cloning

• Recognition sequence is identical on both strands (5’à3’).

• Sticky ends can bind in a complementary fashion.

• Ligases can stick overlapping DNA together.

Vectors

• Vectors carry DNA to the host cell.

• Genetically engineered plasmids.

• Plasmids are found naturally in bacteria.

• Small, circular DNA in the cytoplasm.

• Get copied when bacteria divide.

• Bacteria can make use of the genes carried on the plasmid.

Creating a Vector

• Target DNA and plasmid are digested with the same restriction enzyme (e.g., EcoRI).

• Target DNA has sticky ends.

• DNA recombination occurs using ligase.

• Recombinant vector is formed and introduced into a bacterial cell.

Ligation Outcome

• Plasmid and DNA cut with the same RE.

• Ligation reaction can result in:

  • Plasmid reforms, or joins other plasmids.

  • DNA self-ligates.

  • Correct vector is formed.

Host Cells Replicate the DNA

• Once a vector is produced, it is inserted into a host cell for replication.

• The DNA fragments and the cut plasmid are mixed.

• The sticky ends of different fragments that base-pair are bonded by DNA ligase.

• The result is recombinant plasmids that carry foreign DNA.

• These plasmids are introduced into host cells, which divide to form clones.

Selectable Markers

• Help differentiate between transformed and non-transformed bacteria.

• Usually antibiotic resistance.

• Only transformed cells will grow in the presence of the antibiotic.

Targeting Specific Genes for Cloning

• Many techniques are available to allow targeting specific genes for cloning.

• Fewer clones need to be produced to find the gene of interest.

• Saves time, money, and lab space.

Polymerase Chain Reaction (PCR)

• A better way to copy DNA sequences for study.

• A technique of DNA replication targeting only the DNA you want to study.

• Requires several key components:

  • DNA – something to copy

  • Buffers – to provide the best conditions

  • dNTPs – A’s, C’s, G’s, and T’s to make a new DNA strand

  • Primers – starting point for duplication

  • Taq polymerase – enzyme for copying

Polymerase Chain Reaction Primers

• Short DNA fragments (~20bp).

• Complementary binding to DNA flanking the target sequence.

• Provide a free 3’ end to which Taq polymerase can bind and begin copying.

• One primer per DNA strand.

• 3’ ends of primers must point towards one another.

Polymerase Chain Reaction Taq Polymerase

• Can withstand high temperatures.

• Similar to DNA Polymerase III in our cells.

• Reads DNA underneath and pulls dNTPs out of solution to create a new complementary DNA strand.

• Works in a 5’ to 3’ direction.

Polymerase Chain Reaction – Temperature Cycling

• Temperature cycling is required in PCR:

  • Denaturation

  • Annealing

  • Extension

• Multiple rounds of cycling give exponential increase in your target DNA.

• Quickly builds up lots of copies for further research.

PCR Cycling

• Denaturation – 95°C:

  • Heating the dsDNA breaks hydrogen bonds, resulting in two strands of ssDNA.

• Annealing - 50-65°C:

  • Primers bind in a complementary fashion to ssDNA.

• Extension – 72°C:

  • Taq polymerase binds to the 3’ end of the primers, reads the DNA base on the single strand, and pulls a complementary nucleotide out of the solution.

  • Reforms the double-stranded DNA.

PCR Exponential Amplification

• Following each cycle of denaturation, annealing, and extension, the number of DNA templates doubles, leading to exponential amplification of the target DNA sequence.

PCR and Non-Target DNA

• Target DNA is between the primers.

• Taq polymerase doesn’t know where to stop.

• More and more target DNA is produced over time.

Applications of Cloned Sequences

• Generate animal models for disease:

  • Genetically engineer animals with disease-causing mutations.

  • See what disease processes are occurring.

  • Transfer ideas and findings to human situations.

• Create stem cells for medical treatment:

  • Stem cells have the ability to turn into any cell type or tissue.

  • Used to repair damaged or diseased tissues.

  • Take someone’s own cells to develop treatment.

• Bring back extinct species:

  • Requires DNA from the extinct animal.

  • A closely related species to be an egg donor and surrogate mother.

  • All attempts so far have been unsuccessful.

  • Useful for preserving endangered species.

• Cloning livestock:

  • To get better meat, milk, wool production.

  • Pretty common undertaking.

• Plants to detect environmental changes:

  • When the environment changes, the appearance of the plant changes.

  • Plants can detect landmines.

• Producing drugs:

  • Generate bacteria that will produce human products (e.g., Human Insulin Production).

  • Edible vaccines.

• Cloning pets:

  • With enough money and will, this is possible.

  • Though, they don’t always look the same.

Ethical Issues in Cloning

• Cloning pets:

  • Ethical issues – pet farms.

  • Egg donors, surrogates, unwanted pups/kittens.

• Cloning humans:

  • Ethical, legal, and social concerns.

  • Therapeutic cloning.

Genetic Therapy

• Can we use this technology to correct malfunctioning genes?

• Removing exons with disease-causing mutations, just like the natural process of alternative splicing.

Gene Expression Review

• gene (DNA) -> Pre-mRNA -> mRNA -> protein

• Transcription, splicing, translation, posttranslational modification

What is Exon Skipping?

• A technique that can be used to either:

  • Remove disease-causing exons from the mRNA

  • Skip over additional exons so that the mRNA sequence makes sense

• Masks the exons from the splicing machinery

Maintaining Reading Frame

• mRNA codes for amino acids in 3bp codons.

• A single deletion or insertion moves the codon down one position in the sequence.

• This can completely change the resulting protein produced.

Normal Dystrophin Gene Expression

• Dystrophin gene is on chromosome X.

• Contains 79 exons, ~3,600 amino acids.

• Provides a structural link to maintain muscle integrity.

• Mutations in DMD produce Duchene and Becker Muscular Dystrophies.

Muscular Dystrophy Mutations

• Often a result of skipping or deletion of exons.

• In Becker MD, the reading frame remains intact though the protein may not function as well as it should.

• In Duchenne MD, the reading frame is changed, so No functional protein is produced à more severe.

Exon Skipping

• Artificially skip exons with disease-causing mutations.

• Skip additional exons to return sequence to the correct reading frame.

Sense and Anti-sense

• Two strands of DNA - Sense (coding) and Anti-sense (template)

Anti-sense oligonucleotides

• Anti-sense oligos are identical to the anti-sense (template) strand.

• Cover the splice recognition site, or Bind to exon recognition sites in the exons.

DMD therapy progress

• A number of exon skipping AONs have been produced.

• Show some promising results in studies.

• AONs have been mostly well-tolerated.

• Does not replace lost muscle, but slows progression.

• May be suitable for 83% of DMD sufferers.