AMB 10- Transgenic Organisms

Comprehensive vocabulary flashcards covering the concepts, mechanisms, tools, and regulatory milestones of transgenic organisms and genome editing.

Transgenic Organism

An organism or cell whose genome has been altered by the introduction of one or more foreign DNA sequences originating from another species.

Forward Genetics

An experimental approach that starts with an observed phenotype of interest and seeks to identify and clone the underlying causative gene using techniques such as mutagenesis, QTL mapping, and positional cloning.

Reverse Genetics

An experimental approach that begins with a known gene sequence and investigates its biological function by disrupting or modifying it—via methods such as ectopic expression, gene silencing, gene targeting, or TILLING—and analyzing the resulting phenotypic alteration.

Microinjection

A mechanical transgenic method requiring a micromanipulator and a microcapillary needle to deliver large fragments of linear foreign DNA directly into target cells, such as animal embryos.

Male Pronucleus

The distinct, larger nuclear structure within a fertilized mammalian egg into which foreign linear DNA is injected during the generation of transgenic mice before nuclear fusion.

Biolistics

A transformation method (also known as particle bombardment or gene gun delivery) where microscopic gold or tungsten particles coated with DNA are propelled into target cells or tissues using high-pressure helium gas.

Electroporation

A physical transformation technique in which brief, high-voltage electrical pulses induce a voltage across the cell membrane to form temporary pores, allowing exogenous DNA to enter the cytoplasm before membrane resealing.

Crown Gall

A plant tumor disorder naturally induced by the phytopathogen Agrobacterium tumefaciens through the horizontal transfer and genomic integration of bacterial plasmid DNA into wounded plant cells.

Ti Plasmid

The tumor-inducing extrachromosomal plasmid found in Agrobacterium tumefaciens that carries the transfer DNA (T-DNA), virulence (vir) operons, and genes for opine synthesis and catabolism.

T-DNA (Transfer DNA)

The specific segment of the Ti plasmid, defined by 25-base-pair border repeats, that is excised, transported across bacterial and plant membranes, and integrated into the plant nuclear genome.

Type IV Secretion System

A specialized multiprotein channel complex encoded by the virB operon in Agrobacterium tumefaciens that translocates the single-stranded T-DNA-protein complex across bacterial cell envelopes and into host plant cells.

VirA

A membrane-bound sensory histidine kinase in Agrobacterium tumefaciens that detects wound-derived phenolic signals (acetosyringone, AS) and activates the response regulator VirG via post-translational phosphorylation.

VirG

A transcriptional regulator in Agrobacterium tumefaciens that, upon phosphorylation by VirA, binds promoter regions to induce the transcription of other virulence (vir) operons on the Ti plasmid.

VirD2

A Ti plasmid-encoded site-specific endonuclease and integrase that cleaves the lower strand of T-DNA borders, binds covalently to the $5^\prime$ terminus of the released single-stranded T-DNA, and provides nuclear localization signals (NLSs).

VirE2

A single-stranded DNA-binding protein exported by Agrobacterium that coats the single-stranded T-DNA molecule to protect it from host nucleases, forms membrane channels, and contributes nuclear localization signals (NLSs).

Floral Dipping

An Agrobacterium-mediated transformation procedure used in Arabidopsis thaliana where developing inflorescences are immersed into a bacterial suspension, enabling transformation of female germline tissues without tissue culture.

Agrobacterium rhizogenes

A soil bacterium related to Agrobacterium tumefaciens that carries the root-inducing (Ri) plasmid and is widely utilized in biotechnology to produce hairy root transformations.

Naturally Transgenic Crop

A cultivated plant species whose genome contains functional, expressed Agrobacterium T-DNA sequences acquired through natural horizontal gene transfer during evolution, exemplified by cultivated sweet potato.

Endogenous Retroviruses (ERVs)

Ancient viral DNA sequences integrated into ancestral germlines that comprise roughly 8 to 10 percent of the human genome, occasionally co-opted to produce protective envelope proteins that competitively block modern viral receptors.

CRISPR-Cas9

A microbial adaptive defense endonuclease platform adapted for targeted genome editing, consisting of a Cas9 nuclease paired with a synthetic guide RNA to generate precise double-stranded DNA breaks.

Protospacer Adjacent Motif (PAM)

A short, specific conserved nucleotide sequence directly adjacent to the target genomic site that is required for initial Cas9 binding and activation of endonuclease cleavage.

Non-Homologous End Joining (NHEJ)

An error-prone cellular DNA repair pathway that directly ligates double-strand break ends without an external template, frequently producing random nucleotide insertions or deletions (indels) to disrupt or knock out gene function.

Homology-Directed Repair (HDR)

A precise cellular repair mechanism that utilizes an exogenously supplied homologous donor DNA template to repair Cas9-induced double-stranded breaks, facilitating targeted base substitutions or gene insertions.

Genetic Technology (Precision Breeding) Act

Legislation passed into law in England on 24th March 2023 receiving Royal Assent, removing GMO regulatory hurdles for precision-bred, gene-edited plants and animals that could have arisen via conventional breeding or natural mechanisms.

Casgevy (exagamglogene autotemcel)

A pioneering CRISPR-Cas9 ex vivo gene-editing therapeutic developed by Vertex Pharmaceuticals and CRISPR Therapeutics, authorized in the UK on 16 November 2023 for patients aged 12 and older suffering from sickle-cell disease or transfusion-dependent beta thalassaemia.

• ILO 1- Describe how microinjection, biolistics and electroporation can generate transgenics, including understanding when each technique is most suitable.

MICROINJECTION-

A technique for introducing foreign DNA directly into the nucleus of a cell using a fine glass needle. This method is often used in early embryonic stages to create transgenic animals.

 Micromanipulator and microcapillary needle needed

 - Animals including mice, fish, xenopus, rats, rabbits and large domestic animals such as cattle, sheep and pigs but also smaller microscopic organisms.

 - Large fragments can be inserted

 - Challenging and time-consuming

Making transgenic mice- example

1) Microinject gene, of interest as linear DNA into male pronucleus

2) Introduce several such embryos into pseudopregnant mouse (female mouse whose body hormonal state mimics pregnancy)

3) Birth

4) Somatic cells of offspring tested for presence of gene after biopsy and selected mouse bred to test for gene in germ cells

5) Transgenic mouse

tandemly/ repeatedly arranged copies of the gene are inserted randomly into one chromosome in each cell

• Transgenic mosquitos also made- research this as outside reading

Biolistics-

A method that uses high-velocity microprojectiles to deliver DNA into cells, allowing for the transformation of plant tissues. It is particularly useful for stable transformation of species with tough cell walls.

 Gold or tungsten particles coated in DNA

 - High pressure gas (Helium)

 - Common for plant tissues and some cell lines

 - Transient and stable expression

 Good for plants that are resistant to Agrobacterium-mediated transformation

 - Can transform part of a tissues 

 - DNA inserts randomly

Electroporation-

A technique that uses an electrical field to increase the permeability of the cell membrane, allowing the uptake of foreign DNA. It is suitable for various cell types, including prokaryotic and eukaryotic cells, and can be used for both transient and stable transformations.

 Commonly used

 - Can transform single-celled organisms

 - High voltage created temporary pores

- DNA taken up and integrated into genome

• ILO 2- Explain how agrobacterium-mediated transformation is used to generate transgenic plants.

 Based on natural process (Agrobacterium tumefaciens) – natural plant pathogen

 - Type IV secretion system used by bacteria

 - Tumour inducing plasmid

 - T-DNA is transferred (part of the plasmid)

 Infects many, many species of plants

 - Easy to use

 - Can transform through tissue culture and floral dipping

 - Can transfer large T-DNAs (30kb+)

TI Plasmid and vir genes-

 Not all of the genetic information on the plasmid is transferred into the host genome – only the T-DNA, but the plasmid is needed

 - Several virulence genes, named virA, virB, virC etc.

 - Roles in the transfer of T-DNA from Agrobacterium to its host

 - Can transfer large T-DNAs (30kb +)

Agrobacterium-mediated transformation is a method that exploits the natural ability of the soil bacterium Agrobacterium tumefaciens to transfer T-DNA to plant cells, resulting in stable genetic modification. This technique is widely used for creating transgenic plants due to its efficiency in transferring large segments of DNA and ease of use through methods like tissue culture and floral dipping.

PLANT TRANSFORMATION-

 - Agrobacterium introduced into Arabidopsis using floral dipping

 - Also other methods of plant transformation such as Agrobacterium rhizogenes (hairy root transformation)

TRANSGENIC ORGANISMS IN NATURE-

Sweet potato genome contains evidence of naturally-occurring plant transformation by Agrobacterium

ARE HUMANS TRANSGENIC- they still possess small amounts of viral DNA to protect against infection

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• ILO 3- Demonstrate an understanding of how gene editing technologies fit within the field of transgenics

Gene editing technologies, such as CRISPR-Cas9 and TALENs, allow for precise alterations in an organism's DNA. These techniques enable targeted modifications, enhancing the ability to generate transgenic organisms with specific traits, including disease resistance and improved agricultural yield.

Genome editing-

 Gene expression:

   - overexpression (strong constitutive promoters)

   - conditional expression (inducible promoters)

   - silencing (asRNA /RNAi)

 Gene modification:

   - deletion by Knock Out (KO)

   - replacement with mutant alleles

 CRISPR-CAS9 (type of genome editing)-

-Derived from bacterial enzymes.

-CAS9 and RNA guide.

-‘Molecular scissors’

-

-Efficient and can be used in almost any organisms

-High specificity

• ILO 4- Evaluate challenges in the generation of transgenics, particularly in relation to agriculture, and show an awareness of associated policy issues

Challenges include gene stability, unintentional effects on ecosystems, and regulatory hurdles related to safety and public perception. Issues such as the potential for gene flow to wild relatives and ethical concerns about animal welfare and biodiversity also contribute to the complexity of transgenic development in agriculture.

2018 Court Ruling: The Court of Justice of the European Union (CJEU) ruled in 2018 that all organisms produced via new mutagenesis/gene-editing techniques like CRISPR fall under the 2001 GMO Directive, regardless of whether foreign DNA was added.

The Updated EU Framework - New Genomic Techniques reform: Following recent legislative reforms (including provisional agreements in late 2025), the EU separates New Genomic Techniques (NGTs) into a two-tier system:NGT-1 (Equivalent to conventional breeding): CRISPR-edited plants with small, natural-like modifications (under a set threshold and without foreign/transgenic DNA) are exempt from strict GMO regulations and safety assessments.

NGT-2 (Complex or transgenic modifications): CRISPR edits that involve complex alterations or the insertion of foreign genetic material remain subject to strict GMO rules

But ok in the US, so Atlantic rift!

There is anxiety about the use of transgenics. When CRISPR came along, there was a big hope that CRISPR would be expempt as such specific mutations are introduced that you basically cannot tell the difference. Basically no unwanted mutations.

-Are organisms genetically modified by crispr actually transgenic?-

CRISPR still considered transgenic. Big difference to the States where not only is GM allowed, but CRISPR is considered non-transgenic.

Figure is from a recent paper.  

non-homologous end joining (NHEJ), or homology-directed repair (HRD)

-CREATING TRANSGENICS REQUIRES MULTIPLE DISCIPLINES/ steps

• NEW LAW PASSED- Allowed in England: Food derived from Precision Bred Organisms (PBOs) is legally permitted under the Genetic Technology (Precision Breeding) Act 2023.

• Requires Prior Authorisation: A developer cannot just put a gene-edited food on the market. It must be approved by the Food Standards Agency (FSA) and added to the official public register first.

• Not Allowed in Scotland, Wales, or Northern Ireland: The devolved nations have not adopted these rules, meaning commercial cultivation and sale of gene-edited foods remain restricted there under older GMO frameworks.

• Gene editing has applications in other arras of science e.g. gene editing treatment for blood disorders

Future challenges-

-Many organisms are still difficult to transform

-Efficiency for genome editing can be low

-Improving strategies for generating transgenic plants (precise integrations, copy number etc)

-Appropriate regulation – appropriate understanding needed in policy

-Public perception of genetically engineered plants