UNIT 3: Gene Transfer Methods in Animals and Transgenic Animals

Exam Focus: Microinjection into the pronucleus is the standard technique. Know the definition of a transgenic animal and the specific reasons why transgenic mice are essential to medical research.

Table of Contents

1. Gene Transfer Methods in Animals
   • Microinjection
   • Embryonic Stem Cell (ES cell) Mediated Transfer
   • Retroviral Gene Transfer Method
2. Transgenic Animals
   • Introduction to Transgenic Animals
   • Production of Transgenic Mice

1. Gene Transfer Methods in Animals

The goal is to integrate foreign DNA (transgene) into the host cell's genome, specifically the germline, so the trait can be passed on to subsequent generations.

Microinjection

The most common and efficient method for producing transgenic animals (especially mice).

• **Mechanism:** Purified DNA (the transgene) is physically injected directly into the **pronucleus** (the haploid nucleus of the egg or sperm just before fusion) of a fertilized egg using a fine glass pipette.
• **Result:** The injected DNA randomly integrates into the host chromosome. The egg is then implanted into a surrogate mother.

[Image of pronuclear microinjection]

Embryonic Stem Cell (ES cell) Mediated Transfer

The method of choice for targeted gene modification (e.g., gene knockout or knock-in).

• **Mechanism:** Foreign DNA is introduced into pluripotent ES cells in culture. Cells that successfully incorporate the gene (usually via homologous recombination) are selected. These modified ES cells are then injected into a host blastocyst, which is implanted into a surrogate mother.
• **Result:** Creates a **chimera** (an organism made of two different cell populations), some of whose cells (including germline cells) carry the transgene. The chimera is bred to produce a pure transgenic line.

Retroviral Gene Transfer Method

Uses genetically engineered **retroviruses** (RNA viruses that convert their RNA genome into DNA via reverse transcriptase) as vectors to carry the transgene.

• **Mechanism:** The virus infects the early embryo cell and integrates its DNA cargo (the transgene) into the host genome.
• **Advantage:** High integration efficiency.
• **Disadvantage:** Retroviruses have a size limit for the foreign DNA, and the integration site is random, which can potentially disrupt host genes.

2. Transgenic Animals

Introduction to Transgenic Animals

A transgenic animal is an animal in which an exogenous (foreign) gene, called a **transgene**, has been intentionally introduced and stably integrated into its genome, allowing the transgene to be expressed and inherited by its offspring.

Production of Transgenic Mice

Transgenic mice are the most widely used mammalian model due to their short reproductive cycle, ease of handling, and genetic similarity to humans. They are typically produced using **pronucleus microinjection** or **ES cell-mediated transfer**.