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PTC - C12 (Cell Line Authentication) Cheat Sheet (DRAFT) by

Brief summary of Chapter 12 (Cell Line Authentication) of Plant and Tissue Culture Subject

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Stem Cells

-cells in our bodies which have the ability to differ­entiate into different tissue types
-ability to harness the potential of stem cells to differ­entiate into a specific tissue type will clearly have great benefits in regene­rative medicine
-only the use of epithelial stem cells in skin and cornea grafting, and transp­lan­tation of haemat­opo­ietic stem cells for treating certain blood disorders are clinically establ­ished
-devel­oping in vitro culture protocols and establ­ishing charac­ter­ization standards of stem cells, both embryonic and adult, are therefore vital.

Embryonic Stem Cells

-Human embryonic stem cells (hESCs) are derived from the inner cell masses (ICMs) of blasto­cysts
-They are plurip­otent cells, having the capacity to self-renew and differ­entiate in vitro and in vivo into a wide variety of tissues exhibiting the charac­ter­istics of all three germ layers.
-Pluri­potent, can form 3 layers of ectoderm, mesoderm and endoderm
-Infinite life span due to expression of telomerase
-Form teratoma when injected into immune­-co­mpr­omised mouse
-Clump to form an embryoid with cells that differ­entiate sponta­neously
-Directed differ­ent­iation is possible with addition of specific growth factors that direct cell down a specific pathway of differ­ent­iation
In vivo differ­ent­iation by teratoma formation in SCID mice
-hESCs are capable of differ­ent­iating into tissues of all three germ-l­ayers in vivo.
-This is observed as teratoma formation after transp­lan­tation.
-Inject more than 2 x 10^6 hESCs into a testis of SCID male mouse
-Harvest teratoma after 3-5 months
-Fix teratoma in 4% parafo­rma­ldehyde and embed in paraffin
-Examine teratoma histol­ogi­cally
Teratoma: A type of germ cell tumor that may contain several different types of tissue, such as hair, muscle, and bone. A rare type of tumor that can contain fully developed tissues and organs, including hair, teeth, muscle, and bone. ­Ter­ato­mas are most common in the tailbone, ovaries, and testicles, but can occur elsewhere in the body. ­Ter­ato­mas can appear in newborns, children, or adults. They're more common in females.
SCID: Severe combined immuno­def­iciency

Embryonic Stem Cells Source

Embryonic Stem Cells

Adult Stem Cells

-Undif­fer­ent­iated cells found among differ­ent­iated cell
-Multi­potent, divide to form progenitor or precursor cell e.g. epithelial cell and fibroblast
-Epith­elial cell and fibroblast are prolif­erative in culture, therefore will dominate the culture
-Some are self-r­ene­wable such as hemato­poietic stem cell of bone marrow
-Others are not self-r­ene­wable with finite life span such as mesenc­hymal stem cell
-Fetal stem cell has more potential or capacity for prolif­eration but less adaptable to adult enviro­nment

Normal & Transf­ormed Animal Cells

Normal Animal Cells
Transf­ormed Animal Cells
-Diploid chromosome number with no gross chromo­somal damage
-Chrom­osome fragme­ntation is often associated with transf­orm­ation – aneuploidy
-Anchorage dependent
-May lose anchor­age­-de­pen­dence – cell could grow as suspension
-Finite life span
-Infinite growth capacity – also known as continuous cell line
-Non-m­ali­gnant/ cancerous
-Trans­formed animal cells are not necess­arily cancerous
-Telom­erase activity absent
-Telom­erase activity present
-May lose contact inhibition – cell could grow as multilayer
-Form tumour when injected into immuno­-co­mpr­omised mice
Cancerous cells display malignancy in addition to the charac­ter­istics of transf­ormed cells

Cell Line Identi­fic­ation

-to prevent cross-­con­tam­inated cell being used for research
(googl­e)-­pro­vides resear­chers with confidence that their cell lines are correctly identi­fied, and not cross contam­inated with other cells
1. Karyot­yping
2. Isozyme pattern
3. Antibody labelling
4. DNA finger­pri­nting
5. Telomerase test

1. Karyot­yping

-nucle­opr­otein is partially digested by trypsin, and the Giemsa dye produces a charac­ter­istic pattern of G bands
-banding patterns are charac­ter­istic for each chromosome pair and permit recogn­ition
-banding patterns are charac­ter­istic for each chromosome pair and permit recogn­ition
-deter­mines the species of origin and determine the extent of gross chromo­somal changes in the line
-cell lines with abnormal karyotype are also used if they continue to perform normal function
-Karyotype is affected by the growth conditions used, the way in which the cells are subcul­tured and whether or not the cells are frozen.
-dye gives chromosome a stripped appearance
-it stains the regions of DNA that are rich in adenine (A) and thymine (T) base pairs
-regions that stain as dark G bands replicate late in S phase of cell cycle & contain more condensed chromatin
-regions that stain as light G bands replicate early in S phase & contain less condensed chromatin
1. Incubate hESCs in hESC culture media with 0.1 μg/ml colcemid for 3 hrs at 37°C in a humidified C02 incubator
2. Harvest single cells using trypsi­n-EDTA and treat them with a hypotonic solution (1 % sodium citrate buffer) at 37°C for 30 mins
3. Fix with methanol and acetic acid (3:1,v/v)
4. Spread hESCs onto a glass slide, dry and identify chromo­somes by G banding


2. Isozym­e/I­soe­nzyme Analysis

-based on the existence of enzymes with similar or identical specif­icity, but different molecular structure
-study the patterns of migration of isoenzymes present in cell lysates following electr­oph­oresis using agarose gels under non-de­nat­uring conditions
-patterns obtained are species specific and therefore are used as quality control and authen­tic­ation procedures to confirm species of origin of material
-Specific activity stains are used to develop a banding pattern of isozymes (zymog­ram), which is charac­ter­istic of a particular cell line.
-By using several enzymes the distin­gui­shing features of a cell line are establ­ished.
-These features can often distin­guish cell lines even if derived from the same species.
-This is a more rapid technique compared to karyot­yping and requires smaller cell samples.
-Enzymes: Glucose 6-phos­phate dehydr­ogenase (G6PD), lactate dehydr­ogenase (LDH), nucleoside phosph­ory­lase, alkaline phosph­atase, creatine kinase, glucok­inase, hexoki­nase, glutat­hione S-tran­sferase (GST)

Isozym­e/I­soe­nzyme Analysis

3. Labeled Antibodies

-use of a fluore­sce­nt-­labeled antibody specific for a membrane antigen
-The antibody, is conjugated to a suitable fluore­scent compound, such as fluore­scein.
-The conjugate will bind specif­ically to the outer membrane of the chosen cells which can be identified by fluore­scence microscopy or by a fluore­sce­nce­-ac­tivated cell sorter

Labeled Antibodies

4. DNA Finger­pri­nting

-A unique DNA 'finge­rprint' can be developed for a particular cell line.
-DNA finger­print results from the fragme­ntation pattern produced by digestion of cellular DNA with restri­ction endonu­cleases
-The resulting restri­ction fragment digest is separated by electr­oph­oresis
-Radio­active probes are then used to hybridize to specific restri­ction fragments which can be highli­ghted by autora­dio­graphy.
-results in a charac­ter­istic 'bar-code' pattern
-The most useful probes for this purpose are those that hybridize to 'mini-­sat­ellite' DNA
-These are repetitive nucleotide sequences of varying length found throughout the genomic DNA.
-Certain restri­ction enzymes (for example, Hinfl) are used because they are known to cut DNA within the minisa­tellite regions.
-The length and distri­bution of the resulting minisa­tellite DNA fragments are unique to indivi­duals and hence can be used for identi­fic­ation, including cell line identi­fic­ation.

DNA Finger­pri­nting

5. Telomerase Test

-Initial tumor formation can occur in the absence of telome­rase, tumor mainte­nance requires telomere stabil­iza­tion, most often accomp­lished through the activation of telome­rase.
-If telomerase expression is detected, it is transf­ormed cell line or cancer cell line
-If telomerase expression is not detected, it is deer liver normal cell line
-Telom­erase extend telomeres
-A telomere is the end of a chromo­some. Telomeres are made of repetitive sequences of non-coding DNA that protect the chromosome from damage. Each time a cell divides, the telomeres become shorter. Eventu­ally, the telomeres become so short that the cell can no longer divide.