Table of Contents
Overview
Transferring foreign genetic material means moving DNA or RNA from one organism (or molecule) into another, where it can be maintained and often expressed. In genetic engineering, this is the step that turns an isolated and prepared DNA fragment into a “living experiment” inside a cell or organism.
This chapter focuses on the main experimental strategies for introducing foreign genetic material into:
- Prokaryotic cells (especially bacteria)
- Eukaryotic cells (yeast, plant, and animal cells)
- Whole organisms (e.g., transgenic plants and animals)
You will often see the general term “gene transfer” or “transformation/transfection” used for these processes.
Key Terms
To keep the descriptions short and clear:
- Vector: a DNA molecule (plasmid, virus, artificial chromosome) used to carry foreign DNA into a cell.
- Host: the cell or organism that receives the foreign genetic material.
- Transformation: gene uptake by bacteria and some lower eukaryotes.
- Transfection: gene uptake by eukaryotic cells (especially animal cells) in culture.
- Transgenesis: stable introduction of foreign DNA into the germline so it is inherited by offspring.
- Stable vs. transient:
- Stable: foreign DNA is integrated into the genome or maintained long term.
- Transient: foreign DNA remains for a short time and is then lost.
Natural Gene Transfer Systems and Their Use
Many laboratory methods exploit gene transfer processes that already exist in nature.
Bacterial Transformation
Some bacteria can naturally take up DNA from their environment (natural competence), but in the lab, gene transfer is usually artificially induced:
- Chemical transformation:
- Bacterial cells are treated with salts (often CaCl₂) and chilled.
- Plasmid DNA is added.
- A brief heat shock (e.g., 42 °C for ~30–60 s) increases membrane permeability.
- Some cells take up the plasmid; most do not.
- Transformed cells are selected (for example, using antibiotic resistance encoded by the plasmid).
- Electroporation (bacteria):
- Bacteria and plasmid DNA are mixed.
- A short, high-voltage electrical pulse is applied.
- The electric field opens temporary pores in the membrane.
- DNA enters; pores reseal quickly.
Chemical transformation is simple and cheap; electroporation is often more efficient and can work with strains or species that are hard to transform chemically.
Bacterial Conjugation
Conjugation is a bacteria-to-bacteria DNA transfer through direct contact:
- A donor cell carrying a conjugative plasmid (such as an F plasmid or a modified laboratory “shuttle” plasmid) forms a pilus to connect to a recipient cell.
- A single DNA strand is transferred and replicated, so both cells end up with a complete plasmid.
In genetic engineering, conjugation is useful to:
- Move plasmids from one bacterial strain to another.
- Transfer genetic constructs from E. coli (easy to manipulate) into other bacteria that are difficult to transform directly.
Conjugation is especially important in environmental and industrial microbiology, where target bacteria may not be transformable by simple chemical methods.
Viral Vectors
Viruses are naturally specialized for injecting nucleic acids into cells. Genetic engineering uses modified viruses (viral vectors) to carry desired genes instead of pathogenic genes.
General features:
- Viral genomes are partly deleted to remove harmful functions.
- The gene of interest and necessary control elements are inserted.
- Packaging is often done in “helper” cells that provide viral proteins in trans.
- The resulting viral particles infect target cells and deliver the foreign DNA.
Examples (details of the viruses themselves belong to other chapters):
- Bacteriophages (phages): used to deliver DNA into bacteria, sometimes in place of or in combination with plasmids.
- Lentiviral vectors (from retroviruses): can integrate into the genome of nondividing mammalian cells, useful for long-term expression.
- Adenoviral and AAV vectors: widely used in gene therapy research; often cause mainly transient expression or extrachromosomal maintenance.
Viral vectors can provide very efficient gene delivery, especially into cells that are otherwise hard to transfect.
Physical Methods of DNA Transfer
Physical methods rely on mechanical or physical forces to get DNA across membranes.
Electroporation (Eukaryotic Cells)
The principle is the same as for bacteria, but adapted to eukaryotic cells:
- Cells in suspension are mixed with DNA.
- A rapid electric pulse creates transient pores in the plasma membrane.
- DNA enters the cytoplasm and can reach the nucleus.
Electroporation is used for:
- Mammalian cells in culture.
- Yeast cells.
- Plant protoplasts (plant cells whose cell wall has been removed).
Advantages:
- Works with many cell types.
- Does not require special viral vectors or chemical reagents that might be toxic.
- Can be scaled up for large numbers of cells.
Microinjection
Here, foreign DNA is directly injected into a target cell using a fine glass needle.
Two important uses:
- Injection into single cultured cells:
- DNA is injected into the nucleus or cytoplasm.
- Very precise, but low throughput (one cell at a time).
- Pronuclear microinjection in embryos:
- Common method to generate transgenic animals (especially mice).
- Linearized DNA (often containing a promoter, coding sequence, and selectable marker) is injected into a pronucleus of a fertilized egg.
- The embryo is implanted into a surrogate mother.
- If integration occurs in the germline, the foreign gene is inherited by offspring.
Microinjection gives high control over which cells are modified but is technically demanding and labor-intensive.
Biolistics (Gene Gun)
The gene gun method propels tiny DNA-coated particles into cells or tissues:
- Microscopic particles (typically gold or tungsten) are coated with DNA.
- These particles are accelerated (e.g., by high-pressure gas or a shock wave) toward the target tissue.
- Particles penetrate cell walls and membranes, releasing DNA inside.
Applications:
- Particularly important for plant cells and tissues with strong cell walls.
- Also used in some fungi and animal tissues.
Biolistics can target whole tissues or organs, not just isolated cells, which is useful for plant transformation when preparing protoplasts is difficult.
Laser- and Other Microporation Techniques
More specialized physical methods include:
- Laser poration: very localized, laser-induced membrane openings.
- Ultrasound-based methods: use acoustic energy to transiently permeabilize membranes.
- Mechanical cell squeezing: cells are forced through narrow channels, briefly deforming the membrane.
These are still mainly used in specialized research contexts.
Chemical Methods of DNA Transfer
Chemical methods change the cell membrane properties so DNA can enter.
Calcium Phosphate Precipitation
An older but classic method for mammalian cell transfection:
- DNA is mixed with a calcium salt (e.g., CaCl₂) and phosphate buffer.
- This forms a fine DNA–calcium phosphate precipitate.
- Cells take up these particles by endocytosis.
- Some of the DNA escapes from endosomes into the cytoplasm and eventually reaches the nucleus.
This method is low-cost and can work reasonably well for some cell lines, but efficiency and reproducibility are limited compared to newer methods.
Lipid-Based Transfection (Lipofection)
Lipid-based reagents are now among the most common tools for transferring DNA and RNA into mammalian cells.
Principle:
- Synthetic cationic lipids or lipid-like molecules (liposomes) are mixed with negatively charged nucleic acids.
- They form lipid–DNA (or RNA) complexes.
- These complexes bind to the negatively charged cell membrane.
- Uptake occurs by endocytosis or fusion with the membrane.
- The nucleic acid is released into the cytoplasm and can enter the nucleus.
Uses:
- Delivery of plasmid DNA, siRNA, mRNA, or CRISPR–Cas components.
- Transient or stable transfections, depending on design and selection.
Advantages:
- Relatively gentle on cells.
- No need for expensive equipment.
- Commercial reagents are optimized for many cell types.
Polymer-Based and Other Nonviral Reagents
Besides lipids, there are cationic polymers and other synthetic carriers:
- Polyethylenimine (PEI) and related polymers can condense DNA into small particles.
- Some are designed to help escape from endosomes after uptake.
- Other nanomaterials (e.g., dendrimers, nanoparticles) are also used.
These methods are often aimed at combining efficiency with low toxicity, especially for in vivo and therapeutic applications.
Gene Transfer into Plants
Plant cells pose special challenges: they are surrounded by a rigid cell wall. Key strategies exploit natural plant-associated bacteria or bypass the cell wall.
Agrobacterium-Mediated Transformation
The soil bacterium Agrobacterium tumefaciens naturally transfers DNA into plant cells, causing crown gall disease. Genetic engineering repurposes this system:
Natural process (in brief):
- Agrobacterium carries a tumor-inducing (Ti) plasmid.
- A specific DNA segment of the Ti plasmid, called T-DNA, is transferred to plant cells.
- This T-DNA integrates into the plant genome and expresses genes that form tumors and opines (nutrients for the bacterium).
Engineered process:
- Disease-causing genes in T-DNA are removed.
- The T-DNA region is redesigned to carry genes of interest (plus selectable markers).
- Plant tissues (e.g., leaf discs) are co-cultivated with engineered Agrobacterium.
- T-DNA is transferred and integrated into plant cell genomes.
- Transformed cells are selected and regenerated into whole plants using tissue culture techniques.
This method is widely used for generating transgenic crops, as well as for fundamental research in plant biology.
Protoplast Transformation
To bypass the cell wall:
- Plant cells are treated with enzymes (cellulases, pectinases) to remove their cell walls, producing protoplasts.
- DNA is introduced by:
- Electroporation, or
- Chemical methods (e.g., PEG — polyethylene glycol, which promotes DNA uptake).
After transformation:
- Protoplasts must regenerate their cell wall.
- Under appropriate culture conditions, they can divide and form callus tissue.
- Whole plants can be regenerated from this callus.
Protoplast methods allow relatively direct and versatile gene transfer but require specialized tissue culture expertise.
Biolistic Methods in Plants
As described earlier, the gene gun is especially important for:
- Species or tissues that are hard to transform with Agrobacterium.
- Transformation of chloroplast genomes (plastid transformation), which often requires direct DNA delivery into chloroplasts.
Transformed cells are again selected and regenerated into plants.
Gene Transfer into Animals and Humans
Most methods for animals are aimed at cultured cells (for research and therapy) or at generating transgenic animals.
Transfection of Cultured Animal Cells
Common methods:
- Lipid-based transfection (lipofection).
- Electroporation.
- Polymer-based reagents.
- Viral vectors (e.g., lentivirus, adenovirus, AAV).
Two main outcomes:
- Transient transfection:
- DNA is not integrated into the genome.
- Expression lasts for a limited time (typically days).
- Useful for short experiments (e.g., testing a promoter or protein function).
- Stable transfection:
- DNA integrates into the host genome (randomly or at defined sites).
- Requires selection (e.g., antibiotic resistance) to isolate cells that have integrated the construct.
- Resulting cell lines maintain the foreign DNA over many divisions.
The choice of method depends on the cell type, desired duration of expression, and experimental or therapeutic goals.
Generation of Transgenic Animals
For a foreign gene to be heritable, it must be present in the germline. Several approaches exist:
- Pronuclear microinjection (already mentioned):
- Injection of DNA into the pronucleus of a fertilized egg.
- Integration is usually random; multiple copies can insert.
- Common for mice; more challenging in some other species.
- Embryonic stem (ES) cell methods:
- DNA constructs are introduced into ES cells (usually by electroporation).
- ES cells with the desired genetic modification are selected.
- These modified ES cells are injected into early embryos (blastocysts).
- Resulting offspring can be chimeric, and some gametes carry the modified genome.
- Allows precise gene targeting and the generation of “knockout” or “knock-in” animals.
- Viral transgenesis:
- Viral vectors introduce genes into early embryos or germline cells.
- Integration patterns and copy numbers differ from microinjection.
Transgenic animals are key tools for studying gene function, disease models, and, in some cases, for biotechnological production of proteins.
Somatic Gene Transfer in Gene Therapy
When genetic material is transferred into somatic (non-germline) cells of humans or animals, the goal is usually therapy, not heritable modification.
Typical methods:
- Ex vivo:
- Cells (e.g., hematopoietic stem cells) are taken from a patient.
- Genetic modification occurs in culture (e.g., using lentiviral vectors or electroporation of CRISPR components).
- Modified cells are returned to the patient.
- In vivo:
- Vectors (often viral) are delivered directly into the body (e.g., injection into blood or specific tissues).
- Enable gene transfer into target tissues (e.g., liver, muscle, retina).
Foreign genetic material may:
- Replace or supplement a defective gene.
- Introduce a new function (e.g., in cancer immunotherapy with CAR-T cells).
- Temporarily express therapeutic RNA or proteins.
The specifics of medical applications and safety aspects belong to other chapters, but the key concept here is that the same technical principles of gene transfer are applied in a therapeutic context.
Stable Integration vs. Episomal Maintenance
After entry into the cell, foreign DNA can behave in two main ways:
- Episomal (extrachromosomal):
- DNA remains as a separate molecule (like a plasmid).
- May be replicated if it has appropriate origins of replication.
- Typically leads to transient or semi-stable expression.
- Genomic integration:
- DNA becomes part of a chromosome.
- Occurs by natural recombination processes, sometimes random, sometimes targeted (e.g., by engineered nucleases).
- Enables long-term, often heritable expression.
In practical genetic engineering:
- Plasmid transformation of bacteria usually yields stable, extrachromosomal plasmids.
- Plant and animal transgenesis typically seeks stable integration (except in special cases like viral vectors designed for episomal maintenance).
- Transient transfections are widely used for short-term experiments.
Selection and Screening of Successfully Modified Cells
Because gene transfer is rarely 100% efficient, methods of selecting or identifying successful events are essential.
Common tools:
- Selectable markers:
- Often antibiotic resistance genes (in bacteria) or drug-resistance genes (in eukaryotic cells).
- Only cells that received and express the marker survive in selective medium.
- Reporter genes:
- Encode easily detectable products (e.g., fluorescent proteins, luciferase).
- Allow visual or quantitative detection of gene transfer and expression.
- Molecular tests:
- PCR, hybridization, and sequencing are used to confirm the presence, copy number, and insertion site of foreign DNA.
While these detection methods are discussed in more detail in other chapters (methods of investigation), they are tightly linked to gene transfer: no gene transfer experiment is complete without a way to verify where and how the foreign genetic material ended up.
Summary
- Transfer of foreign genetic material is the central step that turns isolated DNA into a functional construct inside living cells or organisms.
- Natural systems (bacterial transformation, conjugation, Agrobacterium, viruses) are often adapted and controlled for laboratory use.
- Physical methods (electroporation, microinjection, biolistics) and chemical methods (lipid-based and polymer-based transfection, calcium phosphate) provide alternative routes for DNA entry.
- Plants require special strategies due to their cell walls; animals require methods adapted to cultured cells, embryos, or therapeutic contexts.
- Successful gene transfer experiments depend on both efficient delivery and reliable selection/verification of modified cells.
All later applications of genetic engineering—whether in research, agriculture, industry, or medicine—rely on these fundamental methods for moving foreign genetic material into living systems.