Question

4. p53 is an important cancer suppressor gene. To study its function, it is very useful to have p53 knock-out mice. How can one best prepare a strain of p53 knock-out mice? Please start with the optimal method to introduce mutations into DNA, and then describe how you would incorporate such DNA into the mouse genome. List all major steps. please give detailed answer, thank you!

Answer 1

p53 is an important cancer suppressor gene and mutations of the p53 tumour suppressor gene constitute one of the most frequent molecular changes in a wide variety of human cancers.  The functions of the p53 protein include a contribution to G₁ cell cycle arrest to allow DNA repair as well as induction of apoptosis after DNA damage.

At first glance, from a genetic, physiological, and anatomical perspective, however, mice are remarkably similar to humans.

p53 knockout mutant mice develop tumours at three to six months of age. These mice are suitable for use in applications related to the study of familial breast cancers such as Li-Fraumeni syndrome as well as research of lung, brain and bone tumours, lymphoma and leukaemia, and other rare cancers. In 1992, Lawrence Donehower and colleagues achieved the first p53 gene knockout in mice. They used homologous recombination in mouse embryonic stem cells to inactivate the p53 gene. Using molecular biology techniques, these scientists generated two types of mice: heterozygous mice (p53^+/–) that carried only one functional p53 allele, and homozygous null mice (p53^–/–) that lacked both functional p53 alleles and had no functional p53 protein.

The reseachers examined the p53^–/– mice and observed that they developed multiple types of cancerous tissue. These p53^–/– mouse tumor tissue tissues are stained so that acidic components like nucleic acids inside the cell nucleus appear dark purple while other cell and tissue components appear pink.

Making a knockout mouse Step 1: Designing the targeting vector The markers Noo and TK are inserted into the target gene sequence to make a targeting vector sequence Step 2: Inserting the targeting vector into ES cells The targeting vector is inserted into ES cells via electroporation. In some cels, the targeting vector recombines with the target gene and knocks out one copy of the target gene. in other celis, the targeting vector recombines n the wrong place, a random section of the chromosome. Targeting Random Knocked out gene sequence Randem Result cells with knocked out gene are Result cells with random recombinaion are Step 3: Selecting cells Only the cels that have successfully incorporated the targeting vector into the target gene survive in the presence of neomycin and ganciclovir (shown in red Step 4: Injecting cells into a new embryo Cells containing the targeting vector are then selected and injected into a nomal developing mouse embryo Step 5: Breeding The resulting chimeric (spotted) mouse contains a mix of its own cells and the heterozygous knockout cells. This mouse is bred with a normal (white) mouse. Among their offspring are mice that are capable of passing the knocked-out gene to their own offspring

Steps:-

1. scientists design a targeting vector
2. they insert the targeting vector into embryonic stem (ES) cells
3. they select cells that have incorporated the targeting vector into the target gene. The target gene is knocked out in these cells
4. then they inject the selected cells into a normal developing mouse embryo
5. the resulting chimeric mouse is bred to ultimately produce a homozygous knockout mouse.