What are the experimental steps using an ER-Cre/loxP system to trace the Lgr5+ c

Question

What are the experimental steps using an ER-Cre/loxP system to trace the Lgr5+ cells as the intestinal stem cells?

and what is the function of the Lgr5 promoter, tamoxifen and beta galactosidase?

Lgr5 promoter Estrogen-binding domain of the estrogen receptor (ER Made only in cells expressing the intestinal stem cell Lgrgene ER-Cre chimera Cre ER Tamoxifen O Tamoxifen binding moves ER-Cre chimera into nucleus Cytosol Nucleus Reporter gene Splice Splice donor acceptor Blocking Promoter segment-galactosidase loxP loxP ER-Cre chimera ER-Cre chimera removes blocking Blocking segment of DNA -galactosidase expressed in all descendants of this ceü

Explanation / Answer

The Cre LoxP is a recombination thecnique used to study gene inversion,deletion and translocation due to the Cre Lox recombinase enzyme to catatalyze site specific recombination.

in this experiment : The determination of pancreatic beta cell origin

the insulin promoter gene show the expression of tamoxifen dependent Cre recombinase. this enzyme then resist in cytosol as a complex with estrogen receptore(ER) and heat sock protein(HSP). As the action when tamoxifen is injected in the cell the CreER complex will get seperated and Cer recombinase entered the nucleus to form recombination.

now inside the nucleus the Cre recombinase will cutout the LacZ stop sequences and thus allow the expression of HPAP label or short placental alkaline phosphatase.

so by this process cell that normally produce insulin on pancreatic beta cell can be tagged with HPAP .

As we know CRE is used in Linease analysis that is it is used to check the faith or origin of a perticular cell going to transformation.

Ans.2: Lgr5, is a marker of intestinal stem cells, is expressed in actively cycling cells in the bulge and secondary germ of telogen hair follicles and in the lower outer root sheath of anagen hair follicles.It act as selectable marker to identify the transformant.

Tamoxifen is a selective estrogen receptor modulator (SERM).so it is required to seperate CreER complex and Cer recombinase entered the nucleus.

LoxP... its full form is locus of X-over in P1 Bacteriophase.LoxP is a site on P1 bacteriophase consisting of 34bp.They include two systemic 13bp and 8 assymetric bp sequences. because of this assymetry inversion , deletion or translocation is performed by Cre as it combine here in assymetric part.

Related Questions

Navigate

• Browse (All)
• Browse W
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.