Transgenic Mouse Development Sequence

Developing a transgenic mouse involves introducing foreign DNA into its genome, typically at a very early stage of development. One common method is pronuclear microinjection. Let's break down the process and evaluate the given statements to find the correct sequence.

Analyzing Statements for Transgenic Mouse Production

• Statement A: "The transgenic mouse thus born will be a homozygous transgenic animal and can be maintained by crossing with another transgenic animal." This is usually incorrect for the first generation. The injected gene integrates randomly, and the first generation (F0) is typically mosaic or heterozygous. Homozygous lines are established by breeding F0 animals. This statement describes a later outcome and breeding strategy, not a step in the initial development.
• Statement B: "The fertilized transgenic eggs are allowed to develop in vitro." After microinjection, the manipulated eggs (embryos) are indeed cultured in vitro for a short period, usually to the 2-cell or blastocyst stage, before transfer. This is a valid step in the sequence.
• Statement C: "The desired gene is preferably microinjected in male pronucleus after sperm entry in oocyte." Yes, this is the standard procedure for pronuclear microinjection transgenesis. The male pronucleus is usually targeted because it is typically larger and more accessible than the female pronucleus. This happens shortly after fertilization.
• Statement D: "For best efficiency, the desired gene is always microinjected in the male gametes and then they are allowed to fertilize the female gametes." Microinjecting male gametes (sperm) is Sperm-Mediated Gene Transfer (SMGT), a different technique, and pronuclear injection (C) is generally considered more efficient and standard for creating traditional transgenic mice using this method. This statement is not accurate for the described process which involves injecting fertilized eggs.
• Statement E: "Blastocyst stage embryos are transferred to the uterus of hormonally prepared mother." This is a correct and crucial step. Embryos that have developed to the blastocyst stage (or sometimes earlier stages) in vitro are surgically transferred into the reproductive tract of a surrogate mother mouse whose uterus is prepared to receive them.
• Statement F: "The fertilized eggs are collected from specific strain of mouse." Collecting fertilized eggs is a starting point, but statement G provides more detail on how these eggs are obtained for in vitro manipulation (superovulation and IVF).
• Statement G: "The female mouse of specific strain is superovulated, oocytes are collected and allowed to fertilize in vitro." This statement describes the initial steps to obtain a large number of fertilized eggs suitable for microinjection. Superovulation stimulates the female to produce many eggs, which are then collected and fertilized outside the body (in vitro fertilization or IVF). This is a common way to generate the necessary material for microinjection.

Determining the Correct Sequence

Based on the analysis, the standard procedure for creating a transgenic mouse via pronuclear microinjection involves these key steps in order:

1. Obtaining a supply of fertilized eggs. This is often achieved by superovulating females and performing in vitro fertilization (Statement G).
2. Injecting the foreign DNA into the pronucleus (preferably the male pronucleus) of the fertilized egg (Statement C).
3. Culturing the injected embryos in vitro for a short period to check for survival and initial development (Statement B).
4. Transferring the developed embryos (e.g., at the blastocyst stage) into the uterus of a pseudopregnant surrogate mother mouse (Statement E).
5. Allowing the embryos to develop to term and be born.
6. Screening the offspring for the presence of the transgene (identifying transgenic founders).
7. Breeding the transgenic founders to establish stable lines (related to A, but A itself is about the outcome and maintenance).

Comparing this sequence with the options, let's evaluate option 1: G → C → E.

• G: Superovulation, oocyte collection, IVF. (Obtains fertilized eggs/zygotes).
• C: Microinjection into male pronucleus. (Introduces the gene into the zygote).
• E: Transfer blastocyst stage embryos to surrogate. (Places the manipulated embryo for development to birth).

This sequence aligns perfectly with the crucial steps of obtaining material (G), modifying it (C), and allowing it to develop further (E). Statement B (in vitro development) would typically occur between C and E, but it is not included as a separate step in option 1, suggesting the sequence focuses on the major transition points. Statements A, D, and F are either incorrect methods, post-development outcomes, or less complete descriptions compared to G. Therefore, the sequence G → C → E represents the most accurate and logical progression of the major steps involved in developing a transgenic mouse among the provided options.