W5L2 Fri Cellular genetic

Question 1

What is cellular genetics

Answer 1

The study of how the global pattern of gene expression determines the cellular phenotype

Question 2

Attribute that cause the complexity of gene expression

Answer 2

-mRNA level
-isoform
-protein level
-protein isoform
-protein modification

Question 3

Truth about the molecular pathway

Answer 3

-We tend to think linearly. i.e. we pick out simple cause and effect pathways.
-In reality, gene regulatory interactions are massively parallel with extensive cross-talk between pathways.
=To try to predict the effect of changing levels of a gene is difficult due to the many interconnections. The genomics era is forcing us to think in a more “SYSTEMS” level view

Question 4

Ontology meaning

Answer 4

a formal naming and definition of the types, properties, and interrelationships of the entities for a particular domain of discourse

Question 5

The Gene Ontology (GO) project

Answer 5

Founded in 1998, the project began as a collaboration between three model organism databases, FlyBase (Drosophila), the Saccharomyces Genome Database (SGD) and the Mouse Genome Database (MGD).
-The GO project has developed three structured ontologies that describe gene products in terms of their associated:
* biological processes
* cellular components
* molecular functions

Question 6

Detecting GO-term enrichment

Answer 6

-The set of differentially expressed genes after knockout/ knockdown can give us clues about the changes in the cell phenotype.
-look at the function of up/down regulated gene
-If the number of gene affect had similar biological process, it mean something (non random)

Question 7

gene expression in individual cells

Answer 7

-To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) (i.e. hypoblast)
-lineage segregation Ohnishi et al. (2014) carried out qPCR on individual Inner Cell Mass cells.

Question 8

qPCR of 137 cells

Answer 8

-At this early stage only Fgf4 shows a bimodel expression pattern (i.e. cells either have it or they don’t). The other genes are stochastically expressed
-By E4.5, however, the cells have differentiated and are either expressing Fgf4 and Sox2, or Gata6 & Gata4

Question 9

Cell-type discovery by transcriptome profiling of single cells

Answer 9

-Testing expression of genes known to be involved in these cell types was informative.
-Can we profile expression of ALL genes in many cells and deduce common patterns of gene expression?
-might be able to deduce new cell types

Question 10

how to Find cell types using RNAseq

Answer 10

• using antibodies to identify different cell types in the developing lung
  -the AT1 (alveolar type 1) cells express Pdpn, while the
  AT2 (alveolar type 2) cells express Sftpc.

Question 11

Interpreting gene expression arrays

Answer 11

-Heat map for gene expression level
- present of gene product in a cell type
-hierarchal clustering to identify groups of cells that have distinct gene expression pattern
-known marker have an expected patterns

Question 12

Validating new marker genes

Answer 12

-generate antibodies to some of the newly identified gene and confirm if they were found in the same cells as the previously known markers

Question 13

t-SNE: a tool to visualise and discover cell types

Answer 13

-Single cell gene expression data is can be thought of as coordinates where expression levels of each gene give a position along one axis
-Cells of the same type will have similar gene expression and will therefore be close to each other in the N-space

Question 14

Nature of stem cell and medical application

Answer 14

Embryonic stem cells are able to self-renew (so we can culture them in vitro).
They are also pluripotent since they can give rise to the many differentiated cell types of the body (but not the extraembryonic structures).
This has huge potential
in human therapies but there are ethical issues with using human embryos.

Question 15

what is induced induced Pluripotent Stem (iPS) cells

Answer 15

encourage other cell types to express the same genes as epiblast cells could we turn them into pluripotent stem cells

Question 16

Breakthrough of iPS

Answer 16

-four genes (Oct4, Kif4, Sox2 and Myc) which when expressed together in fibroblasts could turn them into pluripotent stem cells.
-Nanog was not necessary, but it forms part of a self-sustaining network in which the genes induce their own and each others expression.

Question 17

In vitro differentiation
of ips

Answer 17

When iPS cells are allowed to form aggregates, they begin to specialise and differentiate.
By supplying the right sequence of signaling proteins and growth factors one can guide them down the differentiation path to defined cell types.

Question 18

induced stem cells, stem-cell therapies

Answer 18

iPS cells hold great promise for treating disease. It will be possible to take cells from a patient, induce them to become iPS cells, repair disease-causing mutations, and then create healthy differentiated cells to reintroduce into the body.

Question 19

Organoid

Answer 19

it has become clear that given the right conditions, some stem cells have the ability to give rise to complex tissues with multiple cell types.
For example, intestinal stem cells (expressing Lgr5) develop organoids that form crypt-like structures, complete with the differentiated cell types such as Paneth cell

Question 20

Organoid applications

Answer 20

-basic research
-disease moddeling
-regenerative medicine