Question

Order the steps in creating a knockout mouse starting at the top.

1) Introduce the interrupted gene into embryonic stem cells, Select for cells containing the marker gene, Inject ES cells containing the knocked-out gene into an embryo early in its development, Disrupt the cloned gene with a marker gene using recombinant DNA techniques, Implant the embryo into a pseudopregnant female
2) Select for cells containing the marker gene, Introduce the interrupted gene into embryonic stem cells, Inject ES cells containing the knocked-out gene into an embryo early in its development, Disrupt the cloned gene with a marker gene using recombinant DNA techniques, Implant the embryo into a pseudopregnant female
3) Inject ES cells containing the knocked-out gene into an embryo early in its development, Introduce the interrupted gene into embryonic stem cells, Select for cells containing the marker gene, Disrupt the cloned gene with a marker gene using recombinant DNA techniques, Implant the embryo into a pseudopregnant female
4) Disrupt the cloned gene with a marker gene using recombinant DNA techniques, Introduce the interrupted gene into embryonic stem cells, Select for cells containing the marker gene, Inject ES cells containing the knocked-out gene into an embryo early in its development, Implant the embryo into a pseudopregnant female
5) Implant the embryo into a pseudopregnant female, Introduce the interrupted gene into embryonic stem cells, Select for cells containing the marker gene, Inject ES cells containing the knocked-out gene into an embryo early in its development, Disrupt the cloned gene with a marker gene using recombinant DNA techniques

Answer 1

To create a knockout mouse, the correct sequence begins with disrupting a cloned gene using recombinant DNA techniques and ends with implanting the modified embryo into a pseudopregnant female.

Step-by-step explanation:

The correct order for creating a knockout mouse involves several meticulous steps in genetic engineering. Here are the steps laid out sequentially:

1. Disrupt the cloned gene with a marker gene using recombinant DNA techniques.
2. Introduce the interrupted gene into embryonic stem cells.
3. Select for cells containing the marker gene.
4. Inject ES cells containing the knocked-out gene into an embryo early in its development.
5. Implant the embryo into a pseudopregnant female.

This process incorporates advanced techniques like CRISPR/Cas editing, the use of reporter genes, and requires screening genetic material through methodologies such as genomic libraries and Southern Blotting.

Answer 2

Creating a knockout mouse involves disrupting the desired gene with a marker gene using recombinant DNA techniques, introducing this gene into embryonic stem cells, selecting cells with the marker gene, injecting these cells into an early-stage embryo, and finally, implanting the embryo into a pseudopregnant female.

Step-by-step explanation:

When creating a knockout mouse, it is important to follow specific steps to ensure that the desired gene is effectively disrupted. These steps are part of a process known as reverse genetics, which is used to understand the function of a particular gene. The correct order of the steps involved in creating a knockout mouse is as follows:

1. Disrupt the cloned gene with a marker gene using recombinant DNA techniques.
2. Introduce the interrupted gene into embryonic stem cells.
3. Select for cells containing the marker gene.
4. Inject ES cells containing the knocked-out gene into an embryo early in its development.
5. Implant the embryo into a pseudopregnant female.

This sequence starts with the use of recombinant DNA techniques to disrupt a specific cloned gene which is then introduced into embryonic stem (ES) cells. Cells containing the knocked-out gene are selected with the help of a marker gene, which enables researchers to identify and separate the modified cells. These manipulated cells are then injected into an early-stage embryo, and the modified embryo is subsequently implanted into a pseudopregnant female mouse where it will continue its development. This process will eventually result in a living mouse with a specific gene knocked out, assisting researchers in studying the gene's function and related phenotypes.