Unit 3 Final Sprint Flashcards
In situ hybridization (when to use, what it is)
Used when you want to see where in an organism a gene is being expressed (create a probe complementary to mRNA you want to study, introduce organism to it, then look at it under a microscope)
Reporter Assay
study signaling pathways in gene regulation (reporter gene, think of it like gfp)
Enhancer
region of DNA bound by activators that increase the likeliness that transcription will occur
operator
What repressive transcription factors bind to (so think of the site lacI binds to)
Enhancer, operators, and promoters are
not part of the DNA sequence (they’re upstream)
When is RNA sequencing used
When you want to measure gene expression of all genes in each sample to see differences.
Necessary Components of a plasmid vector
Origin of replication, terminator, promoter, Amp resistance (selectable marker), gene (cDNA), bacterial promoter
How do we go through bacteria transformation
Cold causes pores in the membrane in which we can insert a plasmid
Random Insertions vs Homologous Recombination
Random Insertion: DNA injected into egg, the transgene is integrated into chromosomes of random nuclei, some transgenic cells from germ line and are passed on Gene Targeting: Embryotic stem cells contain inactive gene and are transferred to embryos (selected via neomycin and ganciclovir)
In a mouse we’re looking for what neo and what ganciclovir
Neo R and Tk - (or no sensitivity to ganciclovir)
How do you make a knockout mouse?
Insert target vector containing neoR and tk into ES of black mouse, Cells are selected by neomycin and ganciclover, only Neo R and no TK survive, Inject ES into the embryo of the brown mouse.
What do hox genes do
Hox genes regulate the identity of body parts (not its structure!)
How do hox proteins work?
Hox proteins are sequence-specific DNA binding proteins and they exert their effects by controlling the expression of genes within developing body parts
What are oncogenes
Proto-oncogenes are genes that normally help cells grow. When a proto-oncogene mutates, it is permanently turned on and causes cell proliferation. (Dominant)
What are tumor suppressor genes
TS genes are genes that slow down cell division, repair DNA mistakes, and tells some cells to undergo apoptosis. When the genes don’t work, this results in proliferation and cancer (inactivated in cancer, recessive)
ras
Ras is an oncogene, a point mutation causes a loss of functionality. It normally cycles between GTP and GDP, but in this case, it can’t cycle from GTP to GDP activating the signal to proliferate.
p53
p53 is a transcription regulator that is activated in response to DNA damage -> no p53 means that the cell goes on without p53 stopping the cell cycle or inducing apoptosis
E2F
E2F is a DNA binding protein that activates expression of S phase genes, Rb binds to E2f and activates at R checkpoint, but Growth factor signaling prevents Rb from binding to E2F by phosphorylating it as a result of Ras and GTP
Familial retinal Blastoma
autosomal dominant
Telomerase
Oncogene, telomerase shortens until cell division stops (telomeres shorten)
XPC
recessive, increased sensitivity to sunlight
Stem Cells Ranked in potency
Totipotency: can produce all cells in an organism: zygote Pluripotency: can produce all body cells in an organism: iPS cells, ES cells Multipotency can produce cells in “organ”: Adult stem cells; Unipotency: Somatic Cells
Stem cells begin differentiation by forming
Progenitor Cells
Stem cells are regulated by
ligands from niche cells
Stem cells
have to touch the niche to be able to renew, otherwise, they go on to differentiate
What controls cell differentiation
regulation of gene expression
Forward genetics
one starts with a question regarding a gene and then proceeds to use mutagenesis to identify the genes involved in the process
question -> gene
Reverse Genetics
one starts with a gene of interest and study its function
gene -> question
___ is epistatic of ____
Ras is epistatic of E2F
