L2-Intro to experimental models & nucleic acid methods

Question 1

Why do we do experiments in Biomed?

Answer 1

-To understand how our bodies work
-To understand how things change in disease
-To find cure or therapies for disease

To understand large things we have to understand a small portion of it first(DNA)

Question 2

How do we do these experiments?

Answer 2

We need to observe, record and analyse.

Question 3

What do we observe to know which methods to use?

Answer 3

DNA
-Restrcition enzyme, cloning, PCR,DNA gels.

RNA
-Q or RT PCR, RNA seq, RNA-ish

Proteins
-Western blotting, Immunohistochemistry, Immunofluorescence, flow cytometry

Question 4

What is an experimental model?

Answer 4

simplified representation or system used in scientific research to investigate and understand complex phenomena. These models are often designed to test hypotheses, explore mechanisms, or simulate scenarios under controlled conditions.

Question 5

What is the purpose of an experimental model?

Answer 5

To create a manageable and replicable environment that allows scientists to isolate variables, measure outcomes, and draw conclusions about the underlying principles of the system being studied.

Question 6

How to reflect what you want to study?

Answer 6

To understand cell cycle protein interactions we can use cells in a test tube. But to understand vaccination and drug outcomes we would nee da animal model or a human model.

Question 7

Factors that can affect the use of experimental models.

Answer 7

1.Accuracy - accurately reflect what you are interested in.
2. Ethics - for animal models, researchers strive to minimise suffering through humane treatment and compliance with regulations.
3. necessity - If you are studying some tissues, cells or proteins that are expressed in the heart, we don’t need to get a full heart from a rat.
If you are trying to understand cell cycle, you don’t need a mouse, we can conduct it in some cells in a test tube.
4. cost - scientific research comes with budgets, so the cost will greatly affect what you have as experiment.
5. Numbers - How many humans are there in UK with the condition you are studying? Are there even enough to get a study?

Question 8

What are some tightly regulated ethics on the use of humans and animals in research?

Answer 8

• All drugs must be tested on animals before humans.
• Use of human tissue itself is tightly regulated.
• you can’t test on animals if there is an alternative. If researchers can achieve the same scientific goals without using animals, they should choose that alternative. This principle aims to minimize the use of animals in research, promoting more humane and ethical science.
• if you do use animals, you will use the smallest one that you can.
  -The use of genetically modified organism is also regulated.

Question 9

Do we need to use a whole organism?

Answer 9

No because the experiments would be much quicker and much more effective. For most basic research you can use cell culture.

Question 10

What are the uses of cell cultures.

Answer 10

To understand cellular strucutres and processes.
ie:-knowing what proteins do allows genetic alteration like in mimicking patient genotypes.

Question 11

Name different types of cell cultures and their pros.

Answer 11

Traditional cell culture > improved modelling of living tissue.
👇
3D culturing > increase complexity
👇
organoids > increased technical difficulty

Question 12

What are the similarities and differences between 2D and 3D cell cultures

Answer 12

Similarities
-Both require appropriate cell lines.
-require the correct cell media.

Differences
-3D cell culture grow cells in a collagen matrix. Cells are floating in collagen.3D cell culture takes collagen which makes up most of us and puts the cells in that.
-More like tissue environment so the cells can move in all different directions. can create gradients. Can change the matrix to be more stiffer or loose depending upon the type of tissue your cells come from.
-3D isn’t always needed.

Question 13

Define Cell line

Answer 13

A cell line is a population of cells that are grown in a laboratory under controlled conditions. These cells are derived from a single original cell and are genetically identical, which makes them a consistent and reliable resource for research.

Question 14

Name the 2 types of cell lines

Answer 14

1. Primary cell line - Take a biopsy directly from a patient, mix it with digesting enzymes and grow it. > can’t grow forever.
2. Immortalised cell line - Gained from the patients in the past but able to grow forever.

Question 15

What should we consider when using a cell line in an experiment?

Answer 15

Google or check what cell media it requires. Because it will be different depending upon which one we use.

Question 16

Define Organoids

Answer 16

-Organoids are 3D cell cultures that mimic the structure and function of real organs.
-From stem cells from an organ >☺can get a variety of different cell types forming.
-Organoids you grow more accurately reflect the actual tissue they come from so need appropriate growth factors and conditions.
-3mm long, no blood supply, no lymphatics, not perfect er

Question 17

Pros of organoids

Answer 17

-creates more than one cell type.
-replicates organs and interactions more accurately
-allows genetic experiments without using an organism.

Question 18

Pros of using single cell organisms.ie:-yeast, bacteria.

Answer 18

-easy to genetically modify. We can use a plasmid or an artificial chromosome with them to produce the DNA or the protein we want.
-easy to grow and to use in the lab.
-cheap.

Question 19

What are they actually useful for?

Answer 19

-producing proteins for your experiments
- simple molecular experiments/interactions (understanding that organism).

Question 20

Pros of using non-human animal models

Answer 20

-helps understand how things work in a complete system.
-gives you massive amount of control. bc they all are genetically identical as they eat the same thing, environment>live the same way and same age. > which reduces the numbers needed for meaningful data.
-can be genetically modified in many cases.
-different animals are used for different questions.
-always uses the smallest model as possible.

Question 21

Examples of animals used for research down the line. biggest to smallest

Answer 21

-Invertebrates - nematodes, Drosophilia Meianogaster, squid.
-Vertebrates that aren’t mamals - Xenopus Laevis, Zebrafish
-Mammals - mice, rats, guinea pigs, farm animals.

Question 22

What are the similarities and differences between mice models and human models ?

Answer 22

S
-genetically similar or identical(ie:-cell lines from humans, mammals)
-very similar on a tissue or organ level.(ie:-animals)

D
-differences are real and you cannot assume things are the same

Question 23

What are the 2 ways of using humans for models ?

Answer 23

1. Clinical trial = testing new drugs/interventions
   2.

Question 24

Why can we use humans for models?

Answer 24

-human volunteers
-tissues - ie: blood, biopsy, whole organs from operations/cadavers.(cell lines will exist)
-can report feelings and outcomes.

But is human work accurate? as variability is the point

Question 25

Why are we not using humans?

Answer 25

-Expensive(millions of £) -must follow animal experiments and safety work -3 key stages : safety, then showing an effect in small and large patient groups. -ethically the most protected species -genetically varied - drug has to work on everybody>genetic variation -genetic modification is almost impossible. -once stratified, very few patients available.

Question 26

It is unethical to have an experiment that gives you meaningless data? How do we prevent that?

Answer 26

-Are working in a model that is relevant. -practical(you'll always do the cheapest accurate thing) -have enough samples (be realistic) -+ve and -ve controls -repeat your experiments

Question 27

How many experiments or repeats to do? and why?

Answer 27

At least 3 repeats -need enough samples so high enough to obtain statistical significance.

Question 28

Why is DNA important in animal modeling?

Answer 28

-To identify the areas of the genome that are important to our work -To modify the genome -To use plasmid as a tool for research

Question 29

Why is RNA important in animal modelling?

Answer 29

- Tells us what DNA is being expressed. - Acts as a proxy/substitute for protein production.

Question 30

Why do we use DNA in the lab?

Answer 30

-basic understanding of genomes -manipulating DNA so it can make what we want. -changing the genome - or adding additional genetic material in.

Question 31

Why do we want to change or edit DNA? 3ways?

Answer 31

Gene therapy -to correct copies of faulty genes in a host organism. - virus vectors. - direct stem cell editing. Genetically modified organisms -transferring genes from different organisms -genes that improve taste/nutritional value or provide resistance to disease can be used to genetically engineer food crops. Sequencing -sequencing small parts of the genome. -fragments of genomic DNA inserted into different "cloning vectors" to allow short lengths of sequencing.

Question 32

Define Recombinant DNA technology

Answer 32

-Experimental methods leading to the transfer of genetic information(DNA) from one organisation to another

Question 33

To perform R.DNA.Tech, we need a lot of copies of Original DNA? What is the technique we use to make copies?

Answer 33

Gene cloning

Question 34

Define clone

Answer 34

a collection of molecules or cells, all identical to an original molecule or cell. ie:- by replicating it inna culture of bacteria.

Question 35

What are the 2 types of genes when gene coding?

Answer 35

Wild type - normal copy of the gene you would find in the wild. Mutant type - altered version of gene with a mutation.

Question 36

Steps for getting the genetic material from the source?

Answer 36

1. 1st step of cloning is to cut out the DNA containing the gene of interest. 2. Restriction endonucleases(from bacteria) cleave DNA in a SEQUENCE-SPECIFIC manner. 3. The evolved as a defence mechanism in bacteria against infection by foreign DNA. 4. Different restriction enzymes in different bacteria. 5. Protect host DNA by modification (methylation).

Question 37

How are type II restriction enzymes named?

Answer 37

They are named after the Genus then species then strain that are found in. then the first endonuclease identified.

Question 38

What do Type II enzymes mostly cut?

Answer 38

palindromic DNA sequence. This is something that reads the same forwards and backwards.

Question 39

Give 2 facts about DNA and palindrome within the genome.

Answer 39

-DNA is double stranded and the strands run antiparallel. -Palindromes are defined as any double stranded DNA in which reading 5'-3' both are the same.

Question 40

What does a restriction enzyme do?

Answer 40

-Each enzyme recognises a 4,6-8 base sequence. -Each enzyme digests(cuts) DNA at a SPECIFIC SEQUENCE.

Question 41

Cut or the cleavage from the type II restriction enzyme

Answer 41

-staggered/sticky ends (A) can be 3' or 5' overhangs OR -can produce "blunt!" ends (B)

Question 42

Name what the DNA ligase can do ?

Answer 42

These can ligate the complimentary ends which are a result of staggered cuts done by an enzyme.

Question 43

How does DNA ligase work with sticky ends? what if they are not complimentary?

Answer 43

-Sticky ends that are complimentary (from digests with the same or different enzymes) can be ligated together. -Sticky ends that are not complimentary cannot be ligated together.

Question 44

How do we make copies of DNA? and how?

Answer 44

Using Plasmids

Question 45

Name features of Plasmid

Answer 45

-naturally occurring extrachromosomal DNA molecules. -small circular dsDNA molecules -range in size from 3-20Kb -easy to purify away from host DNA.

Question 46

What's the relationship between bacteria and plasmid?

Answer 46

-Plasmids can transfer antibiotic resistance from one bacteria to another in the wild. -Bacteria can replicate DNA much better in their cells than we can with PCR.

Question 47

How can we use plasmids can cloning vectors?

Answer 47

-Plasmids are good bc we can make them commercially as cloning vectors. -A cloning vector is a plasmid that can be modified to carry new genes.

Question 48

Plasmids that are useful as cloning vectors must have what ?

Answer 48

-origin of replication>to replicate themselves -selectable marker > resistance gene, such as amp, tet. -multiple cloning site(MCS) >where insertion of foreign DNA will not disrupt replication of inactive essential markers.

Question 49

What do we use to check if the genes are ok?

Answer 49

Primers

Question 50

Facts about using artificial plasmids for cloning.

Answer 50

-A way to screen for your insert -using X-gel containing growth media. -Lacza intact colonies will be blue. -Recombinant white

Question 51

Steps for cloning a gene of interest into a plasmid

Answer 51

1. Cut the gene of interest (gDNA with gene X) using restriction enzyme. 2. Then cut the plasmid using the same restriction enzyme or any restriction enzyme that is compatible. 3. Ligate the DNA fragment with gene X into plasmid (compatible ends) 4. Transfer that plasmid into E.coli and let the those cell grow. 5. Select the right cells with plasmids using LB agar + antibiotic.

Question 52

How to get plasmids into the cells?

Answer 52

-Either transfect them into competent bacteria cells culture or human cell lines.

Question 53

Why do we genetically modify genomic DNA ? examples of how?

Answer 53

-stop genes functioning (knock out) -put in a reporter gene. - deliberately mutate a gene to model a human disease.

Question 54

Name some facts about CRISPR/Cas9 editing.

Answer 54

-Cas9 is a bacterial nuclease>defencing against ds DNA viruses. -Cas9 uses guide RNA to create ds breaks in the DNA>this guide RNA means we can create precise edits. -Cellular DNA repair mechanisms then trim back the DNA. -Normally ds DNA breaks repaired by homologous DNA. -We provide the DNA to be used in the repair. -Can change the gene as we wish.

Question 55

Define reporter genes

Answer 55

To monitor and study the expression of other genes. They produce measurable or visible outputs, such as fluorescence, luminescence, or enzymatic activity, allowing researchers to track whether a particular gene of interest is being expressed.