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CMG francolini > T2/3: Preclinical research models > Flashcards

T2/3: Preclinical research models Flashcards

francollini

1
Q

In vitro vs in vivo studies

A

In vitro: Cell lines, primary cell cultures, 3D cultures, iPSCs, organoids, organs on a chip, organotipic slices
In vivo: animal models

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2
Q

General overview of models in increasing complexity

A
  1. cell culture
  2. tissue culture
  3. cerebral organoid culture
  4. model organisms
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3
Q

What are the advantages of using cell cultures

A
  • allow maintenance of optimal growth conditions for cell viability
    -allow proliferation
    -guarantee maintenance of the characteristics of any specific cell type
    -allow creation of banks for the storage and conservation of cell lines
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4
Q

3 important milestones in history of in vitro cell models

A
  1. Ross Granville Harrison (1907): first in vitro experiment on cultured cells
  2. A lexis Carrel (1912): asepsis
  3. Shinya Yamanaka (2006): discovered iPSCs
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5
Q

Ross granville harrison experiment details

A

Cells were taken derived from the neural tube of a tadpole, placed on a glass depression slide with a drop of frog serum and then the monitored the in vitro development of the nervous fibres from the neural tube.

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6
Q

Alexis Carrel experiment details

A

Development of the culture flask

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7
Q

PROS and CONS of cellular models

A

PROS:
-reduction in the use of animals
-homogeneity within cell lines meaning that observations for one cell hold for all cells
-able to control extracellular environment
-possibility to monitor cell behaviour without changing other factors (as would happen in vivo)

CONS:
-environment is artificial
-behaviour differs from in vivo cells
-low translational power (few deductions can be made)

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8
Q

Types of cell cultures (3)

A
  1. Primary cell culture: cell derived from tissue that can either be pure (one cell type) or mixed (many cell types). Can only divide a limited number of times due to Hayflick’s limit
  2. Secondary cell culture (cell lines): derived from a tumour and so can divide past hayflick’s limit.
  3. Hybridoma: derived from fusion of two cell types and used for production of antibodies
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9
Q

What is the Hayflick’s limit and why does it occur?

A

Def: The number of times a normal cell population divides before entering the senescence phase.

Cause: for each DNA replication there are small sequences of DNA that are not duplicated (telomeres located at the end of chromosomes). Senescence occurs when the telomeres are so short that the DNA cannot replicate and the divisions stop

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10
Q

Role of telomerase in affecting Hayflick’s limit

A

Enzyme which is able to rebuild the sequence of DNA found at the telomeres, hence they don’t shorten during cell divisions.
Consists of a protein and RNA component –> RNA component acts as the template to resynthesize telomeres

!! LEVELS OF TELOMERASE DIFFERS IN DIFFERENT CELL TYPES: not present in somatic cells, reactivated in cancerous cells, and always present in ESCs

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11
Q

Preparation of primary cell cultures

A
  1. tissue is removed from starting organism (usually embryos/larvae)
  2. Mechanical chopping of tissue
  3. Digestion with proteolytic enzymes in aim to achieve complete cell separation
  4. Placement in culture medium
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12
Q

Preparation of hybridoma

A

USED TO PRODUCE MONOCLONAL ANTIBODIES
1.Inject organism (eg. mouse) with specific antigens
2. Triggers B cell and antibody production to that specific antigen
3. B cells extracted from organism
4. B cells placed in vitro to grow alongside myeloma cells (derived from tumour so no Hayflick’s limit of division)
5. Cultured together in the presence of PEG (polyethylene glycol) which induces fusion of the two cell types
6. Hybrid cells present in culture are both able to secrete antibodies AND divide infinitely

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13
Q

Adhesion vs suspension cultures

A

Adhesion: cultivating cells as monolayers on an artificial substrate
Suspension: culturing cells as free-floating aggregates in a culture medium

Cells can grow either in adhesion or suspension depending on characteristics of the tissue from which they come from.

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14
Q

Preparation of tissue cultures

A
  1. Extract desired organ from the starting organism (eg brain)
  2. Sections are placed in different mediums so that they can recover from the stress derived from chopping
  3. Use the sections in desired research

!! tissue cultures are examples of 3D cultures

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15
Q

Description of organoid culture

A

3D highly differentiated systems derived from stem cells. Can be replicated by induced proliferation and differentiation of stem cells.

Tissue is harvested from starting organism, dissociated into fractional units, enriched with stem cells and placed in culture medium with floating spheroids added.

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16
Q

Uses of an organoid culture

A

-MODEL DISEASES and study development of desired organ

SOS: Stem cells used to create organoids can be taken from patients –> hence the culture can analyse morphology and responses to drugs that are patient specific

-allows the study of organs without affecting patients
-higher complexity than cell and tissue cultures which allow study of whole pathway systems/cell collaboration rather than individual cells

!! higher the complexity of the culture, higher complexity of info deduced and studied

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17
Q

Model organisms, definition and reasons for usage

A

-non human species that are widely studied and used in lab to understand biological processes
-usually organisms that are easy to maintain and breed
-embryos might be particularly robust and allow the study of development
-might occupy an advantageous position in the evolutionary tree that gives the research high translational power

!!! ability to share data and replicate experiments within the scientific community where the same model organisms are used

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18
Q

Characteristics of model organisms that make them advantageous in genetic and molecular research

A

-easily able to breed in labs
-short generation time (efficient when studying inheritance patterns)
-easy to mutate (to study characteristics of genetic disease)
-similarity of genome to humans

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19
Q

Characteristics of model organisms that make them advantageous in research of human diseases

A

-possession of mutated orthologues (correspondent genes) between humans pathologies and models
-high level of conservation between human and model genome (due to orthologues being present on similar lengths of the chromosomes)

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20
Q

animal model examples for this course (4)

A
  1. nematode worm
  2. fruit fly
  3. zebrafish
  4. mouse
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21
Q

C. elegans

A

NEMATODE WORM:
- transparent worm: able to study organs, development and process of internal fertilisation
-worms are either male or hermaphrodite
-entire genome was published
-does not have a circulatory system or bones but shares many genes with humans
-grown cheaply
-cultures can be frozen and defrosted when needed
-apoptosis can be studied because it is identical to human apoptosis
-allows CELL LINEAGE: developmental history of a tissue or organ from a fertilized embryo

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22
Q

drosophila melanogaster

A

FRUIT FLY:
-mainly used for inheritance or mutations because its genome has been mapped
-shares 75% of disease-causing genes with humans
-have POLYTENE CHROMOSOMES: oversized with multiple filaments, hence they are easier to see - used for study of chromosome deletion and rearrangements
-easily mutated (with X-ray or chemicals) to create transgenic organisms

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23
Q

Danio rerio

A

ZEBRAFISH:
-transparent, used to study internal organs
-produce hundreds of embryos per mating, fertilization is possible every day of the year and occurs externally
-share 70% of genes with humans
-vertebrate, hence contains same organ systems as humans
-easy manipulation of embryos because fertilisation is external
-mainly used for pharmacological or toxicological screenings

DISADVANTAGE: longer generation time (months to get from larvae to adult)

24
Q

Mus musculus

A

MOUSE:
-used to simulate human genetic disorders, study new therapies
-modeling of diseases that occur in both mice and humans with similar molecular pathways
-SCID mice are used to study tumour cell proliferation and infectious diseases
-ability of genetic modification

25
Q

Creation of transgenic mice for research

A
  1. portion of a plasmid DNA containing desired gene is microinjected into the male nucleus of a fertilised egg
  2. implantation of transgenic zygote into surrogate female
  3. F1 mice produced are tested for the transgenic gene (eg. DNA analysis from a small portion of the tail)
  4. Transgenic positive organisms are selected and used in research
26
Q

Knock-in, Knock-out procedure in mice

A

-genetically modified embryonic stem cells are transferred to a blastocyst
-Implantation of transgenic blastocyst into a surrogate female
-F1 generation contains some individual mice that are chimeric
-Selection of chimeric mice and use in research (expression/lack of transgenic gene expression can be observed through PCR)

27
Q

3Rs in animal welfare

A

Replace: replace animal studies with other methods

Reduce: use as few animal trials as possible

Refine: refine procedures to minimise animal stress

28
Q

Regulations on hESC use in italy

A

-It is forbidden to use hESCs derived within own country, but it is possible to use
hESCs from other countries
-Max 3 embryos implanted for IVF
-Excess blastocysts generated in the past cannot be used to obtain ESCs (hence it is impossible to generate new ES cell lines)

29
Q

Discovery of cloning experiment and conclusion

A

Gurdon experiment,1968
- Used frog skin cells and tadpole gut epithelial cells which were both cultured
-An unfertilized egg was treated with UV radiation to destroy its nucleus
-Insertion of either the nucleus from the cultured skin/epithelial cells
-Creation of clone of whichever cell’s nucleus was implanted

CONCLUSIONS:
1. demonstrated that a fully differentiate cell has the same genotype as a pluripotent cell
2. demonstrated that a differentiated cell can be brought back to stem state

30
Q

Cloning of the first mammal experiment

A

Dolly the sheep, 1996
-uses somatic cell nuclear transfer (SCNT)
-same approach used by gurdon was used: transfers a nucleus of a somatic cell into an enucleated oocyte
-fusion of the egg and of the somatic cell nucleus occurred using a small electroshock.
-zygote became an embryo and was put back into the mother sheep uterus

!First example of reproductive cloning (because it results in the cloning of an entire organism)

31
Q

why is reproductive cloning (like SCNT) a very inefficient process

A

The ratio of somatic cells obtained and used in the SCNT method vs the number of successful organisms that were cloned was very low. There is also a large loss of cells during the procedure which is why it cannot be used with humans

32
Q

Difference between reproductive and therapeutic cloning

A
  1. Reproductive Cloning: to generate an identical copy of an entire organism
  2. Therapeutic Cloning: to generate a blastocyst to obtain (human embryonic) stem cells to be used as a therapy
33
Q

Therapeutic cloning process and advantages

A

Process: the nucleus is donated by the patient needing treatment, and the egg is donated by any random donor (any organism). hESC cells from the inner cell mass of the blastocyst would be obtained during development and these cells would show pluripotency. This can be used in therapy

ADVANTAGES: patient’s immune system will not face rejection as this will resemble an autologous transplant

34
Q

Discovery of iPSCs details

A

Shinya Yamanaka in 2006
-Showed that a differentiated fibroblast grown in a culture with a cocktail of specific factors can revert back into its undifferentiated state and exhibit pluripotency. (hence it can differentiate into any cell of the 3 germ layers)
-These factors (named the yamanaka factors) were established by creating knockout cells each missing 1 of the 24 factors considered and observing which ones prevented pluripotency from being achieved
-Yamanaka then had to prove pluripotency using the assays seen in the unit (not a chimera because these are hESCs)

35
Q

Four Yamanaka factors + pathway systems

A
  1. LIF pathway activates the yamanaka factors Myc and Klf4
  2. Exact pathway for nanog is unknown
  3. WNT frizzled pathway activates the yamanaka factor Sox2
36
Q

Advantages and disadvantages of using iPSCs

A

ADVANTAGES: no ethical controversies, can be differentiated into human cell types that cant be obtained in other ways, used to model human diseases, used to model single gene mutation diseases, can be genetically manipulated and re-implanted within patient

DISADVANTAGES: can be subject to genetic abnormalities (point mutations, chromosome translocation, changes to karyotype), possibly tumorigenic

37
Q

Main applications of iPSCs

A
  1. model disease
  2. screening
  3. drug testing
38
Q

Different strategies to program iPSCs:

A
  1. DNA transfer:
    -viral vectors
    -transient transfection
  2. DNA FREE transfer
    -mRNA
    -miRNA (microRNA)
    -Recombinant proteins
39
Q

VIRAL VECTORS as a method to induce iPSCs

A

Split into the use of retroviruses, lentiviruses and sendai virus/adenovirus

  1. RETROVIRUS:
    Advantages: integration of genome within host genome, high gene expression, they can be easily silenced
    Disadvantages: Poor reprogramming efficiency
  2. LENTIVIRUS:
    Advantages: integration of genome within the host genome, mutagenesis /cancer increased risk, high reprogramming efficiency
    Disadvantages: they cannot be easily silenced
  3. SENDAI/ADENOVIRUS:
    Advantages: high reprogramming efficiency, absent from reprogrammed cells
    Disadvantages: Costs
40
Q

TRANSIENT TRANSFECTION as a method to induce iPSCs

A

Advantages: no reprogramming without genomic integration of the plasmid DNA, no innate antiviral responses
Disadvantages: occasionally genomic integration may occur which can cause mutagenesis, poor reprogramming efficiency

41
Q

mRNA method to induce iPSCs

A

Advantages: reprogramming without genomic integration of exogenous DNA, fast reprogramming, no innate antiviral responses
Disadvantage: mRNAs released from cells can trigger an inflammatory response, cells have to be transfected daily for up to 2 weeks, reduced cell survival after transfection, high cost

42
Q

miRNA as a method to induce iPSCs

A

Advantages: reprogramming without genomic integration of exogenous DNA, fast reprogramming, no innate antiviral responses
Disadvantage: cells have to be transfected daily for up to two weeks, reduced efficiency, high cost

43
Q

RECOMBINANT PROTEINS as a method to induce iPSCs

A

Advantages:
Reprogramming without genomic integration of exogenous DNA, no innate antiviral responses, potential use as a therapeutic agent
Disadvantages: purity and quality of recombinant proteins, time:56 days needed in vitro, high cost, works well with cells from newborn but inefficient with cells from adult

44
Q

saccharomyces cerevisiae as an animal model

A

-small unicellular eukaryotic
-1.25-2 hour replication time
-easy to genetically manipulate (addition and deletion of genes)

45
Q

Ways to create disease in an animal model for experimental use

A
  1. spontaneous change models: diseases analogous to human conditions that occur naturally within organism being studied
  2. Induced disease models: disease triggered by an environmental change that causes organism stress (eg. exposure to drugs, hormones, dark/light/electrical stimuli)
  3. Genetically modified organism model: insertion of disease causing gene/triggering of gene mutation causing defect
46
Q

A&D of spontaneous disease models

A

A: good for immunotherapeutic studies, more relevant to clinical treatments (eg. formation of tumours developing in a way that better resembles natural course of formation in humans)

D: low chance that the disease will develop, hard to maintain in the process and waste of resources if disease doesn’t develop, expensive to maintain, takes a long time

!!!negligible use in research

47
Q

Types of tests performed on an animal model

A
  1. behavioral tests (response to stimulus)
  2. EEG analysis (electroencephalogram)
  3. Respiratory functions
  4. (f)MRI analysis (structural alterations)
48
Q

Transgenic model definition

A

Animals in which one or more genes are modifies, deleted or added.

49
Q

Process of creation of a transgenic animal model

A

AIM: transgene (desired gene) inserted into DNA of germinal cell lines so future generations inherit it

  1. Desired gene and its promotor region (marks initiation of transcription) are attached to a reporter gene (marker that allows identification of whether the altered gene has been taken up by organism in the final stage)
  2. Transfection of vector plasmid into host organism: stable transfection when it is incorporated into genome, and transient transfection when vector remains external
50
Q

Position effect in vector insertion def

A

Risk of insertion occurring in an area that is important for organism survival - coding regions

51
Q

3 ways to insert a transgenic gene into an organism

A
  1. DNA microinjection
  2. retrovirus-mediated
  3. ESC mediated
52
Q

process of vector transfection via ESCs

A
  • DNA with desired vector is introduced into ES cells
    -ES cells injected into early mouse embryo (blastocyst)
    -incorporation into cells of inner cell mass
    -injection into pseudopregnant mouse
    ADVANTAGES: low risk of position effect due to screening of ES prior to injection. rapid method
    DISADVANTAGES: F1 chimera, not all species have available ES
53
Q

process of vector transfection via retrovirus

A

-host cells infected with retrovirus (containing genetic info as RNA)
-incubation in vitro to allow infection
-implantation in a recipient female
ADVANTAGES: high percentage of embryo survival and requires small number of animals
DISADVANTAGES: use of a virus increases risk, generates F1 chimeric mice (multiple breeding cycles are needed for complete gene expression)

54
Q

pseudopregnant recipient/surrogate animal definition

A

female previously mated with vasectomised male so it will activate the necessary hormonal system for pregnancy

55
Q

knock out gene tech def

A

deletion/silencing of a gene to cause loss of gene function

56
Q

constitutive vs conditional kock out tech def

A

constitutive: target gene permanently inactivated in whole animal
conditional: inducible inactivation of gene expression takes place as tissue specific/ temporal manner

CMG francolini (7 decks)

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