Hypoxia models

Question 1

Explain the adaptation strategy of barheaded geese to low oxygen environment ?

Answer 1

The barheaded goose has a substitution of proline at position 119 for alanine at position 119 in the alpha subunit of hemoglobin. This subtitution allows for the equilibrium to change more towards the R (relaxed) state, which results in higher oxygen affinity.

Question 2

What are the 3 functions of hemoglobin ?

Answer 2

Oxygen transport
Carbon dioxide transport
Buffering

Question 3

What is the general structure of hemoglobin ?

Answer 3

Hemoglobin is a protein consisting of 4 subunits, with each subunit containing a heme group and a globin protein.

Question 4

Describe the oxygen and carbon dioxide transport functions of hemoglobin.

Answer 4

Oxygen : hemoglobin binds to oxygen in the lungs where oxygen concentration is high. It then releases oxygen in the tissues.
Carbon dioxide : Transports carbon dioxide, a waste product of cellular respiration from the tissues back to the lungs.

Question 5

Explain the lack of hemoglobin in antarctic icefishes ?

Answer 5

These fish lack hemoglobin for their environment is very rich in oxygen (cold water). Their metabolic rate is also lower, reducing their oxygen needs. Their heart size is increased to pump more blood and is accompanied by higher cardiac output. Blood volume as well as capillaries size is increased.

Question 6

How does a luciferase assay work ?

Answer 6

A luciferase assay measures the activity of luciferase, an enzyme that produces bioluminescence. It is paired with a promoter gene that comes before.

Question 7

What are the main steps of a plasmid isolation protocol ?

Answer 7

1. Cell harvesting
2. Cell lysis
3. Clearing the lysate
4. Binding of plasmid DNA
5. Washing
6. Elution

Question 8

What is the point of lysing the cells ?

Answer 8

The adding of buffers breaks open the bacterial cells, releasing the plasmid DNA in the solution.

Question 9

What is the point of clearing the lysate ?

Answer 9

We clear the lysate by centrifuging the debris (pellet), leaving the plasmid DNA in the supernatant.

Question 10

What does the binding of plasmid DNA step do ?

Answer 10

It binds the plasmid DNA onto the silica membrane. Once the column is washed, it will remove contaminants but the plasmid DNA will remain bound to the silica membrane.

Question 11

What does an Immunoprecipitation do ?

Answer 11

IP is a technique used to isolate specific proteins or protein complexes from a complex mixture based on an antigen-antibody interaction.

Question 12

What’s the difference between transduction, transfection and transformation ?

Answer 12

Transduction : Adeno or lentivirus, permanent
Transfection : Cells, use of plasmids, 48-72hrs because lysosomes eventually get rid of plasmid
Transformation : bacteria

Question 13

How can an immunoprecipitation be used to observe the interaction of 2 proteins ?

Answer 13

Co-IP : an antibody that specifically recognizes one of the proteins is incubated alongside the protein of interest. Agarose beads are added to bind to the antibody-protein complex. Elution to recuperate bound proteins and any associated partners. Analysis with Western blot or mass spectrometry.

Question 14

If an overexpessed transcription factor is found, how can we observe its effect?

Answer 14

By using knockouts or knockdowns with CRISPR-Cas9, transduction, transformation, transfection, TALEN…

Question 15

What are the advantages and disadvantages of CRISPR-Cas9 ?

Answer 15

It’s a permanent change but lots of offtargets mutations can be caused. It is however generally safe.

Question 16

How do you overexpress a TF in a cell ?

Answer 16

Knock-in (in vivo) or transfection

Question 17

What does B-ME do ?

Answer 17

It deactivates RNAse

Question 18

What does a qPCR do ?

Answer 18

It quantifies the amount of DNA or RNA in a sample using a fluorescent probe.

Question 19

What are the advantages and disadvanges of SYBR green probe for qPCR ?

Answer 19

Advantages : SYBR Green is versatile as it can be used for quantifying any PCR-amplified DNA target without the need for specific probes.
Cost effective.

Disadvantages : SYBR green can bind to any dsDNA, including non specific PCR products, giving false positives.

Question 20

What are the advantages and disadvanges of taqman probe for qPCR ?

Answer 20

Advantages : Taqman probes are highly specific. Taqman only binds to specific sequences.

Disadvantages : Expensive, complex to design.

Question 21

Loading for qPCR :

Answer 21

Glut1 and VEGFA = HIF1alpha
EPO and EPAS1 = HIF2alpha
CYBG and PHD3 = Both in HIF1 and HIF2
SHDA is the housekeeping gene

Question 22

HIF (hypoxia inducible factor) pathway ?

Answer 22

Under normoxic conditions :
1. PHD (prolyl hydroxylase domain) enzymes hydroxylate HIF-alpha
2. The hydroxylated HIF-alpha is recognized by the VHL (von hippel lindau) protein, leading to ubiquitination and subsequent degredation of HIF-alpha
3. As a result, HIF-alhpa levels remain low and HIF target genes are not activated

Under hypoxic conditions :
1. PHD enzymes is inhibited due to lack of oxygen, a necessary co-factor for their enzymatic activity
2. As a result, HIF-alpha is not hydroxylated and escapes recognition by VHL
3. HIF-alpha accumulates and translocates to the nucleus where it dimerizes with HIF-1beta
4. The HIF-alpha/HIF-beta complex activates the transcription of target genes
5. These target genes are involved in angiogenesis, erythropoiesis etc.

Question 23

What do restriction enzymes do ?

Answer 23

Restriction enzymes precisely cut DNA at specific sites allowing for the insertion of genetic material in plasmid vectors.

Question 24

What is a miniprep ?

Answer 24

Miniprep, short for mini plasmid preparation is a procedure used to isolate plasmid DNA from bacterial cells.

Question 25

What are the steps of the project ?

Answer 25

1. Prepare the plasmid constructs (Promoter only, KIE, LIE and KIE + LIE) by designing primers used for PCR amplification of those regularatory regions. Also select a vector containing the luciferase reporter gene and a MCS upstream. 2. Ligate the amplified regions into vectors upstream of the luciferase reporter gene. 3. Transform the ligated plasmid into E.Coli cells in proper medium. 4. Mini-prep to recuperate Plasmid DNA. 5. Transfect plasmids into HEK293T cells under both normoxic and hypoxic (0.2%) conditions for 24hrs. 6. Luciferase reporter assay

Question 26

Why do we transform the plasmid into E.coli cells ?

Answer 26

It provides a high yield of plasmid DNA for e.coli rapidly replicates the plasmid DNA. The initial ligation contains a very small amount of plasmid DNA.

Question 27

What does the glut1 cycle do?

Answer 27

It serves for the transportation of glucose across the plasma membranes of cells.

Question 28

Why is a renilla luciferase plasmid transfected as well ?

Answer 28

It serves as an internal control. The dual-luciferase assay system allows for the measurement of both Firefly and renilla activities. The ratio of firefly luciferase activity is thus normalized in each sample. This ensures observations are due to experimental conditions and not due to differences in transfection efficiency

Question 29

What are the roles of HIF1-alpha and HIF2-alpha ?

Answer 29

HIF1-alpha : Response to acute hypoxia Angiogenesis Cell survival HIF2-alpha : Response to chronic hypoxia Erythropoiesis Iron metabolism Tumor growth and metastasis

Question 30

What are Kelly cells ?

Answer 30

Kelly cells are a human neuroblastoma cell line. They are known to activate HIF and to provoke angiogenesis. It thus makes sense to observe a higher expression of VEGFA (vascular endothelial growth factor) in Kelly cells for they want to metastasize.

Question 31

What are 786-0 cells ?

Answer 31

They're a human renal carcinoma cell line

Question 32

What is the link between gene GLUT1 and hypoxia ?

Answer 32

Glucose transporter 1 is a gene involved in cellular glucose uptake. The upregulation of GLUT1 expression in hypoxic cells enhances glucose uptake, allowing cells to maintain energy production through glycolysis even in the absence of sufficient oxygen. (HIF1-alpha)

Question 33

What is the link between gene PAI1/Serpine1 and hypoxia ?

Answer 33

Serpine1 is involved in the inhibition of the fibrinolytic system, that is the breakdown of blood clots. Also linked to angiogenesis.

Question 34

Why are different constructs used ?

Answer 34

Each construct represents a different portion of the EPO gene's regulatory region. Various constructs can help us pinpoint which specific regions are responsibles for EPO expression.

Question 35

What is the promoter construct ?

Answer 35

Its the contruct containing just the promoter region of the EPO gene.

Question 36

What is a housekeeping gene used for in a qPCR ?

Answer 36

It serves as an interal control or regerence gene to normalize gene expression data.