Week 6: Gene Modification in Plants, Animals and Humans

Why can it bad for prokaryotes to carry out gene cloning?

Prokaryotes don’t have the ability to glycosylate proteins

Why is glycosylation important for cloning eukaryotic genes?

• Glycosylation – 50% of eukaryotic proteins are glycosylated

• Carbohydrates are added to proteins as co- and post-translational modifications. N-linked and O-linked

• Glycosylation can affect protein function and protein-protein interactions, protein uptake, secretion, half-life, solubility and antigenicity

• N-linked glycosylation is common in eukaryotes but not in prokaryotes! Bacteria are not capable of proper glycosylation of recombinant mammalian proteins and may need to use other systems

What can be used instead of prokaryotes for gene cloning?

• Higher organisms may be better suited for protein production based on yield required, posttranslational modifications

• also, we can use genetic engineering to improve/cure them→ more complicated systems

Examples of genetically modified organisms

1. bacteria

2. mammalian cells

3. yeast→ hard to extract proteins

4. algae

5. crop plants

6. livestock

Why do we need to deliver genes/ DNA into cells?

Many of cells do not have uptake mechanisms such as bacteria

Name some delivery methods

• Electroporation→ electricity punctures holes in cell membrane- hard not to kill cells

• Chemical treatment

• Biolistics→ take DNA and fire it into cells very fast

• Microinjection

Issues with random insertion into genomes

• unpredictable levels of expression

• insertional mutagenesis

• may cause gene in genome to be expressed when they shouldn’t be, could disrupt expression of important genes

• Gene does not normally confer a selective advantage to host (usually opposite!)

  • Point mutations

  • Rearrangements

  • DNA methylation

How will organisms try to suppress the expression of recombinant genes

• Bacteria sequesters unwanted proteins

• Higher organisms shuts down expression → methylation, point mutations, rearrangements

What are promoters?

• Sequence of DNA where proteins e.g TFs bind to initiate transcription

• organism specific

• Often need spatial or temporal control

  • Inducible promoters (time)

  • Tissue-specific promoters (place) → e.g rice - seeds not leaves?

Tet Off

• tetracycline response element (TRE) driving our gene, and it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter

• the presence of tetracycline, the tTA transactivator goes off, and there is no expression.

Tet ON

• While in a Tet-Off system, tTA is capable of binding the operator only if not bound to tetracycline or one of its derivatives, such as doxycycline

• in a Tet-On system, the rtTA protein is capable of binding the operator only if bound by a tetracycline

Expression control system in bacteria

Lac operon

Why are plants useful for genome modification?

• All cells are totipotent

• Can de-differentiate to recreate a new plant

• Possible to alter just one cell and recreate an altered plant

• If we can deliver DNA to plant cells, we will generate a whole GM plant

What is the biggest issue with plants?

how to get DNA into those cells

How was DNA initially inserted in plants?

• used Biolistic method (gene gun)

• Directly ‘fire’ DNA into cells

  • Originally a CO2-powered Crosman air pistol!

  • Now helium-propelled tungsten/gold particles

What technology is used to insert DNA into plants more recently?

• Agrobacterium biology

• Agrobacterium tumefaciens

How does agrobacterium biology work?

• plants can become infected by Agrobacterium → causes tumours by manipulating the plant → Uses horizontal gene transfer (T-plasmid) to cause tumours in plants

  = (Crown gall disease)

• Transfers ‘T-DNA’ (15-30kb) to cause dysregulated growth

  • T-DNA encodes for auxins

  • Opines for selective growth advantage

  • Integrates into plant’s genome

Why is agrobacterium especially helpful for plants?

• Agrobacterium contains a DNA sequence that allows integration into plant genome

• Can be altered to include specific genes

  • Antibiotic resistance

  • increased growth

  • plant promoters (ie. nos)

How could you engineer a plant to be resistant to roundup?

• Selection experiment → find a plant resistant

• Identify an enzyme that breaks it down—> see what glyphosate targets (aa biosynthesis). Targets EPSPS

• Bacteria which grow in the production plant of roundup

How is herbicide-resistant rapeseed (and soybean, cotton and maize) engineered?

• Herbicide-resistant rapeseed (also soybean, cotton & maize)

• Resistant to glyphosate (Roundup)

  • 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) - aa biosynthesis -

  • Bacterial resistance enzymes added to genome

• More pesticides can be used

• Gene transfer to weeds = resistant weeds?

How can corn be given natural immunity to insects?

Insecticide-containing:

Bacillus thuringiensis proteins

Delta-endotoxins, pore-forming (in insect cells, kills)

• clone gene for delta-endotoxins

• integrate into corn

• natural immunity to insects (e.g corn borers)

  • Less insecticide needed

  • Meant to be inactive on mammals

  • Possibly off target effects..butterflies, bees.

How can rice be GM?

• Vitamin A deficiency kills 700,000 children under 5 a year due to poor diet

• Golden rice → Oryza sativa rice engineered to produce 23x beta-carotene (vitamin A) in the grain

• 2 years of ‘successful’ field testing

• rice is missing 4 enzymes to produce vitamin A

• certain organisms can reduce 4 enzymes to 1→ CRTI

• Phytoene synthase (starts biosynthetic pathway) found in maize

• Glu promoters

• Pmi → positive selective marker

• T plasmid right border, left border

How is senescence shown in tomatoes?

• Accompanied by cell wall degradation

• Accompanied by increase in N- glycoproteins

• After ripening

How can the shelf-life of tomatoes be extended?

• large change in glycosylation of proteins found

• inhibition of enzymes through the expression of RNA interference (RNA i)→ binds to RNA→ causes RNA to become ds, and it decays

• (more detail) Inhibition of N-glycoprotein modifying enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex)

• depleted RNA → extended life

• Longer lasting tomatoes, bananas, strawberries

• Can be ripened for longer in the sun

If you didn’t know what caused senescence in fruit, how would you try to extend its shelf life?

1. look at natural variation

2. mutant tomatoes

3. protein differences across time

4. differences in protein modifications

5. look at gene expression across life

6. identify correlation→ changes correlating with senescence process→ changes may be causative (drivers of change or consequences)

What is genetic modification used for in animals?

• Food production/quality (increased yield, parasite and pest resistance)

• Therapeutic applications (GH, organ transplant)

• Humanised products (breast milk)

• Research: Knockout/knock-in models

How does GM in animals differ to GM in bacteria and plants?

• Bacteria are clonal and plants cells are totipotent

• Most cells in adult animals have limited potency

• Alter every cell in adult? Too difficult!

• It is possible to use a skin cell to make a transgenic animals → nucleus into enucleated embryo like dolly→ very inefficient

• Alter developing embryo

• We want altered germline

• can breed/cross offspring

Cell development in animals

• Cells lose potential as development progresses

• Zygote is first stage in development→ truly totipotent

• Pronuclear phase:

  • First sign of fertilisation

  • Both nuclear membranes dissolve

  • Haploid genomes combine

  • Permissive to foreign DNA incorporation

How can we deliver DNA into the egg?

Pronuclear Microinjection

• Glass micropipette with 0.5um diameter

• +200x magnification

• Negative pressure micromanipulators

• Steady hands!

Technique very inefficient and difficult

How can you make a transgenic animal?

1. Embryonic stem cells taken/ isolated from blastocyst stage

2. Introduced DNA recombines in place of original gene in rare cases

3. Randomly incorporated/ in specific locus

4. Select for successful recombination via resistance marker (heterozygous)

5. Transfer into an enucleated oocyte- incorporated into inner cell mass

6. Transfer to surrogate mouse

7. Breed to create homozygous mutant

The target cell maybe 1 out of 128 so not a clone

The mouse = chimera -mix of GM cells and normal

What is a useful property of early embryonic cells?

• Early Embryo stem cells are highly pluripotent not totipotent

• Can convert cells later in development to early, pluripotent embryonic stem cell like state

• Cells incubated in culture and continue to proliferate

How can you make an animal clone?

• GM cells -sperm/ egg/ germline + cross with another mouse

• 1st gen = chimera

• 2nd = clone

What can gene targeting constructs be used for?

• Add selection/reporter gene

  • Important as correct incorporation is a rare event!

  • Disrupt/alter/insert gene(s) e.g. frameshift, deletions, insertions, gene additions, reporters etc

How does homologous recombination work?

• targeting vector contains sequences that are known to be part of the genome which flank the piece of DNA that we want to insert into the genome

• targeting DNA will go into the nucleus + pairs up with the region of DNA that has homologous sequences

• Recombination can occur from early development

• Product of recombination event: endogenous DNA as in the DNA in the genome in between the target sequences will be replaced by the DNA of interest

• Put in a selectable marker in between those two targeting sequences and have essentially a big deletion of DNA from the genome in between → deletion of genome → loss of function

• Higher organisms are diploid → disrupt once copy doesn’t mean the other is disrupted

• Breed two heterozygous → homozygous → gene knockout

What system can you use to generate conditional knockouts?

Cre/Lox recombination system

How does the Cre/Lox recombination system work?

Cre recombinase recombines Lox sequences

• Generate mouse line with gene of interest altered by adding flanking LoxP sites (Floxed mouse)

• Breed with mouse that expresses Cre recombinase (in next generation the gene will be gone -floxed out!-)

• Can provide deletions, insertions, translocations and inversions

• Cre recombinase can be expressed in particular tissues to provide selective knockouts

How can you control the expression of Cre?

• In bacteria - inducible expression system

• put Cre under the control of tetracycline

• remove in a specific tissue→ use a tissue specific promoter

Example of GM animal: Enviropig

• pigs are bad at removing phytate→ excretion → phosphorous in runoff→ algal blooms

• Pig can produces more environmentally-friendly faeces

• Phytase production in saliva

• Mouse secretory promoter

• E.coli phytase gene to break down phytate

• Less phosphorous run-off

• Reduces algae overgrowth and subsequent anoxia of rivers/lakes (eutrophication)

Example of GM product: Humanised cow milk

• Breast milk replacement

• Pronuclear injection of human genes that improve immune function:

  • Lysozyme

  • Lactoferrin

• 20-30% increased fat content, stronger taste

Vectors for bacteria

• plasmids

• bacteriophages

Transduction

transfer of DNA through viral infection

What can transduced cells be used for?

can then be cultured, used for nuclear transfer cloning or adult cell transplantation and gene therapy

Common features of viral vectors

• Multiple cloning site

• Selection gene(s)

• Reporter gene(s)

• Promoters

  • Ubiquitous

  • Tissue specific

• Enhancers (WRPE) to increase expression

How can you make viral replication incompetent?

• Deletion of essential viral components

• Use of separate vectors for packaging and viral production

How can you make it host specific?

• production of viruses/viral vectors with foreign viral envelope proteins.

• Generates a pseudotyped virus particle

Promoters for expression in any cell

• reporters/ markers

• ‘ubiquitous’ promoters

• preferences for different cell types

Tissue specific promoters

• Relies on expression of tissue-specific transcription factors

• Aimed to be unique to particular tissue

• Examples:

  • Myosin for muscle

  • Cytokeratin 18 for skin

  • ApoE for liver

Promoters with high expression levels

CMV

• commonly used in most cells lines (HeLa, HEK293, HT1080)

MSCV

• hematopoietic and stem cells

Promoters with medium expression levels

EF1 and PGK

• robust in most cells types, primary cells + stem cells

Promoters with low expression levels

UbC

• low and steady in most cell types, primary ells and stem cells

What kind of vectors are adenoviral vectors?

Non-integrating DNA virus

• Not integrated into genome and not replicated during cell division

• Hijacks cellular transcription/translation to make viral proteins

• Highly immunogenic as involved in human respiratory diseases→ make high levels of proteins

• Usually used for in vitro

• protein production experiments

What kind of vectors are retroviral vectors?

Integrating RNA viruses

• Use reverse transcription to integrate RNA into genome

• Inserted DNA is replicated during cell division

• Random insertion points can cause cancer: insertional mutagenesis

• Can cause random integration event

What kind of vectors are lentiviral vectors?

Subset of retroviruses

• integrate into genome via reverse transcription + integrase (enzyme)

• KEY: Can infect non-dividing cells

  • Terminally differentiated cells

  • Neurons, liver cells

  • Stem cells

Example of lentiviral vectors?

HIV-based vectors

• Self inactivating

• Replication incompetent

• Currently on 3^rd generation

What do we have to do to viral vectors before delivering them?

• Essential packaging and replication elements are transfected into same cell separately

  • No complete HIV virus is produced as a vector!

  • Produced virus only contains our insert – cannot replicate new virus in new cell

  • Packaging envelope can be pseudotyped for particular cells and immune reactions (VSV-G infects most cell types)

What effect does ethical considerations have on delivery strategies?

No germline modifications allowed so no pronuclear transfer of blastocysts (or somatic cells!) – now permitted in certain cases for research purpose

In-vivo virus delivery direct to patient

Very immunogenic option – especially adenovirus (Jesse Gelsinger)

• Many different insertion points in different cells: insertional mutagenesis

• In some cases needs to be inserted in particular cell

  • Cystic fibrosis: cystic fibrosis transmembrane conductance regulator (CFTR) in lung/intestine

When can carrier cells be used for deliver?

• When systemic release is sufficient

• diabetes, factor viii

When can exo-vivo transduction of transplantable cells be used?

stem cells

• in vitro differentiated embryonic stem cells - controversial

• Induced pluripotent stem cells – safety concerns

• Adult (somatic stem cells) – often hard to culture

Cautionary tail for gene therapy

• Jesse Gelsinger had a genetic disease called ornithine transcarbamylase (OTC) deficiency

• OTC deficiency prevents the body from breaking down ammonia, a metabolic waste product

• Patients need a strict non-protein diet to control OTC deficiency

• Gelsinger volunteered for a gene therapy experiment based on mouse model observations in which a vector carrying a normal OTC gene was injected into his liver.

• The vector being used to deliver the OTC gene was adenovirus, but he had an immune reaction to the injection, and four days later, on September 17, 1999, he died.

Somatic stem cells

Tissue-specific cells that regenerate tissues throughout life

• can self-renew

• can repopulate whole tissue

• can be taken from patients themselves e.g bone marrow stem cells

Ex vivo transduction of stem cells

1. Normal gene isolated from healthy subject

2. Gene is clones

3. Gene is inserted into retrovirus vector

4. Bone marrow sample taken from patient with genetic defect

5. Marrow cells are infected with retrovirus

6. Transfected cells re-infused into patient

7. Patient observed for expression of normal gene

Advantages of ex-vivo transduction of stem cells

• Self-renewal means clonal growth of transduced cells

• Allows screening/selection of transduced cells

• Reduces ultimate immunogenicity/toxicity (no direct virus)

Gene therapy for severe combined immunodeficiency (SCID)

• Many forms of disease that results in T- and B-cell dysfunction, 2 most common are:

  • X- linked Cytokine gene mutation (IL-2)

  • Defective adenosine deaminase (ADA)

• Early bone marrow transplant can cure in some cases

• 17 children cured so far based on transplant of cells with corrected genes

  • 2 reversions to defective ADA: no selective advantage à silenced

  • 4 leukaemias due to insertional mutagenesis

Gene Therapy Success Story: treating (degenerative) forms of inherited blindness

• The retina, on the inside of the eye, is both easy to access and partially protected from the immune system.

• Viruses can't move from the eye to other places in the body

• Most gene-therapy vectors used in the eye are based on AAV (adeno-associated virus).

• In one small trial of patients with a form of degenerative blindness called LCA (Leber congenital amaurosis), gene therapy greatly improved vision for at least a few years (RPE65). However, the treatment did not stop the retina from continuing to degenerate.

• In another trial, 6 out of 9 patients with the degenerative disease choroideremia had improved vision after a virus was used to deliver a functional REP1 gene.

Gene targeting

• Use CRISPR/Cas9 system to induce

• ds DNA breaks and correct via homologous recombination in cultured stem cells

Zinc Finger Nucleases

• Artificial combination of zinc finger DNA binding domain to DNA cleavage recognition sequence

• Can be engineered to cleave DNA at specific sites

  • Operates as a dimer

  • 9-18bp recognition sequences possible

  • Relies on subsequent recombination