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Transgenic Techniques

🔬 Transgenic Techniques

Modern Approaches to Genetic Modification and Gene Editing

Transgenic techniques have been used for a number of essential scientific goals: to determine an unknown gene’s function, to analyze the malfunction of a mutated gene, to model human diseases, and to develop enhanced agricultural and pharmaceutical products through the creation of transgenic plants and animals.

For example, insect-resistant transgenic plants have been successfully engineered. While the benefits of genetically modified organisms (GMOs) are wide-ranging, debate continues around the ethics of altering plants and animals, as well as the environmental impact of such modifications.

🧬 Methods of Transgene Introduction

There are several ways to introduce a transgene into an organism. Among the most common techniques:

💉 Microinjection

Microinjection is the direct injection of a transgene into the nucleus of a fertilized egg, where it may randomly integrate into the host genome. Introduced in 1981, this method is especially prevalent in the production of transgenic mice.

Once injected, the egg is transferred into a surrogate mother. If successful, the resulting organism will carry the transgene in every cell.

🧬 Retroviral and Transposable Element Insertion

Two other methods for random gene insertion are:

  • Retroviral insertion, which integrates genetic material using viral vectors.
  • Transposon-based systems, which use mobile DNA elements to insert genes at different sites in the genome.

These systems are often used in embryonic development, stem cells, and model organisms.

🔧 Gene Targeting and Knockout Organisms

A major advance in transgenic technology in the 1990s was the development of gene targeting, particularly the production of "knockout" organisms. A knockout refers to the deliberate disruption of a gene, so that it no longer produces a functional protein.

This is achieved by:

  • Replacing a functional gene with a mutated transgene
  • Using homologous recombination to insert the disrupted gene into the chromosome

This allows scientists to study what happens when a specific gene is missing from the organism.

🧫 Embryonic Stem Cell Techniques in Transgenics

In mice, transgenes can also be introduced into embryonic stem (ES) cells, which are then injected into early embryos. The embryos become mosaic organisms—containing both modified and unmodified cells.

If the altered cells contribute to the germline, their offspring will inherit the transgene. Breeding these mice can result in homozygous knockouts, where both gene copies are altered. This allows scientists to study the biological role of the targeted gene in detail.

This knockout technique is widely used in:

  • Mice
  • Yeast
  • Insects

🧬 Knock-in Mutations and Conditional Gene Expression

🔄 Knock-in

The knock-in technique replaces a gene with a mutant or altered version, allowing researchers to:

  • Study gene variants
  • Observe changes in gene function
  • Understand redundant gene pathways

🔄 Conditional Mutation

Conditional mutation techniques allow researchers to:

  • Turn a gene on or off
  • Control when and where a gene is expressed
  • Study developmental and tissue-specific functions of genes

These systems often use inducible promoters or site-specific recombination systems like Cre/loxP.

🧬 CRISPR/Cas9 and TALENs in Transgenic Engineering

🔬 CRISPR/Cas9 & TALENs

More recently, TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 systems have become essential tools in transgenics and genome editing.

They are used across various organisms and cell types including:

  • Mice, rats, zebrafish, Drosophila, C. elegans
  • Human stem cells and induced pluripotent stem cells (iPSCs)

⚙️ How They Work:

  • CRISPR/Cas9 uses RNA guides to direct the Cas9 protein to specific DNA locations for cutting and editing.
  • TALENs use customizable DNA-binding proteins to target and modify specific gene sequences.

These tools enable:

  • Precise knockouts
  • Knock-ins of disease-relevant mutations
  • Correction of defective genes
  • In vivo genome engineering

CRISPR/Cas9 and TALENs have significantly advanced the ability to treat the root cause of genetic conditions, enabling applications not feasible with traditional gene therapy.