Manipulating the Genome and Developmental Commitment

Manipulating the Genome and Developmental Commitment

A colleague’s laboratory has discovered multiple genetic mediators of congenital heart disease via high-throughput sequencing and bioinformatics analyses. Their work suggests that genetic modifications of thePTPN11gene might be an important risk factor for a subset of patients. You would like to generate a genetically modified Ptpn11“knock-out” mouse to studyits role in heart development.Here is the gene model for Ptpn11adapted from NCBI (https://www.ncbi.nlm.nih.gov/gene/19247)…

(3 points) Draw aconcisely annotated diagram of a “targeting vector” that could be used to generate a “knock-out” that does not express a functional Ptpn11gene (diagram must be of your own making, do not snip or copy/paste from elsewhere). Your vector should include components that ensure the mutant mouse will be generated via homologous recombination and NOT non-homologous recombination.

(3 points) You finally have a small tube containing your targeting vector plasmid. In a few sentences, describe the typical process of generating a gene-targeted mouse with the targeting vector from A. What experiment would you perform to confirm the loss of PTPN11 protein in a mouse model?

I would use GFP to confirm the loss of PTPN11 by observing to see if this gene will fluorescence under a microscope. I would also use

(2 points) In a separate set of experiments, you are pleased to discover that Myh7bis specifically expressed in the mouse heart by embryonic day 10 (E10) (see figure to right).In the same week, you also find your Ptpn11^-/-embryos exhibit lethality by E5. You are now unable to study heart formation in this model. Given your recent discovery regarding Myh7b, briefly describe an alternative method you might use to “conditionally knock-out” the Ptpn11gene only in the heart? Be sure to describe how you will determine whether Ptpn11loss has occurred specifically in the heart and NOT in other organ systems, such as the lungs.

(2 points) A collaborating lab has discovered major anatomical defects in the heart of a Ptpn11^D61G/+mouse (heterozygous expression of an Asp61Gly (D61G) point mutation in Ptpn11). Briefly explain how would you test whether “determination” of Ptpn11^D61G/+cardiac muscle cells has occurred during embryogenesis?