MMG115 Midterm 2

for uc davis mmg115 with s. namekawa and l. xu

How can E. coli be transformed to take up foreign DNA?

• chemical treatment using divalent cations and heat shock

• electroporation

What are the steps to transforming E. coli by chemical treatment?

1. Treatment with divalent cations (i.e. Ca²⁺, Rb²⁺). They neutralize the negative charge on the membrane.

2. Treat with a heat shock to generate thermal imbalance on the two sides of the cell membrane. Only a small fraction of cells actually transform.

3. Transformed cells are grown in growth medium for a short time without selection before being plated onto a selection agar plate.

How do selective markers on a plasmid work?

Plasmids have selective markers such as antibiotic resistance genes that can help us confirm if we grew competent and transformed cells.

What are the steps to transforming E. coli by electroporation?

1. introduce a short but strong electric pulse to temporarily open tiny pores'

   • treating cells with high voltage for a short period of time to make cells porous. DNA can enter directly through bacterial cell

What are the different E. coli strains you could possible use depending on your scientific purpose?

• E. coli strain containing deletion in the 5’ region of the lacZ gene to allow alpha complementation using pUC-like vectors

  • allows for blue/white screening

• E. coli strain containing mutations in the hsdRMS system (which encodes EcoK type 1 restriction system).

  • removing a system that could lyse our foreign DNA

How do you select an screen for the recombinant clones

1. antibiotic selection for clones acquiring plasmid

2. screening for clones aquiring the correct recombinants

How does antibiotic selection work?

1. Multiple types of antibiotic selection

2. some inhibit cell wall synthesis, some inhibit protein synthesis

How do we find the correct recombinant clones?

• not all plasmids we use will use the blue/white screening because you need the multiple cloning site (MCS) in the lacZ gene

1. blue/white color screening

2. isolate DNA and perform diagnostic digestion

3. isolate DNA and perform diagnostic PCR

4. isolate DNA and sequence the junction of vector-insert

How does blue/white screening work?

Transformed bacteria are plated onto agar plate containing X-gal and ampicillin

Blue colonies = non-recombinant (can break down X-gal)

White colonies = recombinant ( cannot break down X-gal)

Alkaline Lysis Method

method to isolate plasmid dna from bacteria cells

1. use NaOH and SDS to lyse E. coli

   1. NaOH denatures DNA, including plasmid and genomic DNA. since plasmid is circular, they stay interlinked, but the two strands of genomic DNA are separated from each other and become large pieces of single stranded DNA with nicks

2. potassium acetate is used to neutralize alkaline

   1. the plasmid DNA quickly renatures because they are interlinked

   2. the genomic DNA stays single stranded and precipitates out of solution because they can’t find their exact partners again. ssDNA havr low solubility

3. potassium acetate also reacts with SDS to form KDS, which is cell debris of ss genomic DNA.

what is left is plasmid DNA in salt solution

Plasmid preparation using DNA-binding columns

separating the plasmid dna from salts

• clarified lysate has optimal pH and salt concentration for DNA to bind matrix of silica on a column

• wash the column with salts and ethanol to remove the contaminants

• high salt with low pH causes DNA to bind to the beats, low salt with high pH causes DNA to elute

• elute with Tris buffer

Diagnostic restriction digestion

method to screen for recombinant clones

empty vectors are the plasmid backbone and only has one RE site, so it cuts once into linear DNA

recombinant vectors bring in a new striction site that the empty vector didn’t have

• if we use two different restriction enzymes, cut one in and out of recombinant DNA, we can examine the product to see which one is the right recombinant clone

• recombinant has 2 RE sites, so it will cut into fragments

screen for recombinants by DRD, then follow with electrophoresis of these products

Gel electrophoresis

migration rates of macromolecules thorugh porous matrix in electric field

influenced by:

1. charge of molecule

2. shape of molecule

3. size of molecule

migration rate (if all are the same size):
supercoiled > linear > circular

charge on nucleic acids

nucleic acids at neutral pH are uniformly negatively charged, one unit of negative charge due to the phosphate group per nucleotide

EtBr staining

• Ethidium Bromide is intercalating agent used as a fluorescent dye for nucleic acid gel electrophoeresis

• inserted between stacking bases

• EtBr is also a mutagen, it can cause mutations in DNA

SYBR Green staining

• binds to minor groove of dsDNA

• fluoresces under blue light

• more sensitive and less toxic

• used for nucleic acid detection in real time PCR

steps to performing agarose gel electrophoresis of DNA

1. dissolve agarose pwoder in buffer

2. let agatose gel solidify/polymerize

3. load dna samples

4. apply current to pull dna towards positive electrode / anode

5. dna fragments begin to separate based on size

6. stain gel and visual under UV light

relationship between linear dsDNA migration rate and molecular weight

linear dsDNA migrates at a rate that is inversely proportional to the log of its molecular weight

diagnostic restriction digestion fragments with gel electrophoeresis

• cut backbone does not have the insert, the smaller cut travels further

• the cut ligation product has an insert, and the larger insert carries our gene of interest, so it will be heavily and does not travel as far.

• both have the same linearized back bone length that travelled

principles of sanger dna sequencing

add ddNTPs during dna synthesis instead of dNTPs, which prevents the DNA chain from extending by DNA Polymerase

• template of unknown sequence → add dna polymerase, four dNTPs, and a small amount of fluorescently labeled ddNTPs

• denature and now you have dye labeled segments of dna that is copied from template with unknown sequence

• these segments are applies to capillary gel and subjected to electrophoresis (remember small molecules travel faster and pass through the detector first)

secondary and tertiary structure of RNA

linked base pairs with each other

diff RNA of same sizes can form completely different structures

need to remove these structures / need to remove base pairing

denaturing RNA structures

• glyoxal (prevents base pairing)

• formamide or formaldehyde (disrupts hydrogen bonding between bases)

steps to performing gel electrophoeresis of rna

1. dissolve agarose pwoder in buffer

2. let agarose gel solidify/polymerize

3. denature rna with glyoxal or formamide/formaldehyde and then load rna samples

4. apply current to pull rna towards positive electrode / anode

5. rna fragments begin to separate based on size

6. stain gel and visual under UV light

gel electrophoresis of protein

• protein are not uniformly charged because some carry pos, some carry neg, and some carry neutral charge

• protein are also not uniformly shaped

therefore to get an accurate representation of protein, they need to be linearized and the only depending factor is the size of the molecule

protein denaturation by SDS

SDS has a hydrophobic and hydrophilic end

• hydrophilic end gives negative charge to the molecule

• hydrophobic part binds with the amino acid

when SDS is added it disrupts non covalent interactions (not covalent ones, including disulfide bonds)

• protein denatures, and on average every two a.a. binds one SDS molecule

what we get is a denature protein that carries uniform charge relative to its length

original method of determining dna sequence

sanger sequencing: approx. 700 base pairs per read

next generation sequencing

• billions of reads generated in parallel

• enables rapid, large scale genome sequencing

• cheaper than sanger sequencing

applications of next generation sequencing

• non-invasive prenatal testing

  • examples cell free DNA from maternal blood sample to catch any genetic defects

• cancer detection

• disease risk assessment

• diagnosis of rare genetic diseases

DNA library

a collection of DNA fragments

examples:

• genomic libraries

• cDNA libraries

• next generation sequencing libraries

genomic libraries

collection of cloned DNA fragments representing an organism’s genome

• overlapping fragments allow us to reconstruct the original genomic sequence

• scientists used to sequence genomes by cloning dna fragments and then sequencing them by using sanger sequencing, and then assembling the overlapping sequences

practical problems/issues with genomic libraries

complete digestion with a single Restriction enzyme made some fragments too small or large for the chosen vector. The adjacent fragments also do not overlap, so genome assembly was difficult.

solution to genomic libraries due to restriction enzymes

rather than using restriction enzymes, we do random fragmentation by mechanical shearing

• dna molecules break at different/random positions

  • generates overlapping fragments for genome assembly

problems with cloning large dna fragments

• larger plasmids more difficult to clone and maintain

• lower transformation efficiency than smaller ones

problem: we want to construct genomic libraries with large dna inserts to minimize the number of clones required to cover the whole genome

options for large dna inserts

1. bacteriophage lambda based vectors

   1. among earliest vector used for genomic libraries

   2. insert capacity: up to 20 kb

   3. require phage packaging and infection steps

2. bacterial artificial chromosomes (BACs)

   1. insert capacity: 100-300 kb

   2. used for cloning and and maintaining large DNA fragments

   3. can be handled in bacteria much like standard plasmids

bacterial artificial chromosomes

• plasmid based vector that maintains large dna fragments in bacteria

• publicly availible bac libraries allow us to obtain genomic fragments of interest

• prepare dna probes for fluorescence in situ hybridization

• make transgenic animals

cDNA libraries

colection fo dna copies of mRNAs expressed in a particular cell or tissue

cDNA is made from mRNA using reverse transcriptase

cDNA fragments are typically cloned into plasmid vectors

we use cDNA libraries comparing the cDNA sequence with the genes encoding them

• useful way to identify 5’ to 3’ transcript ends and the introns and exons

genomic vs cDNA libraries

in genomic library creation, you start with the chromosomal dna and fragment it by mechanical sheering. now you have overlapping dna clones in genomic dna library

in cDNA library, you have the same chromosomal dna and you transcribe the genes present on that dna

• both transcripts are then spliced and introns are removed

• if one gene has more transcription than another then you can determine the expression and frequency of expression

• i.e. for every 1 clone of A you make, you make 5 clones of B

more genomic vs. cdna libraries

genomic libraries:

• represent entire genome

• same across cells/tissues of same organism because the genome is the same regardless of hte cell

• gene expression levels do not affect clone representaiton

• this included introns, prmoters and intergenic regions

cDNA libraries

• represent expressed mRNAs

• differ among cells/tissues: if you make a cDNA library from brain vs skin cell, they will be different because gene expression is different for both of them

• gene expression levels affect clone representation

• include exons and UTRs, but generally lack introns and promoters because they are spliced out

next generation sequencing library preparation (short read)

1. fragment the DNA

2. add adapters to both ends of every fragment

   1. the adapters are the sane for all fragments (but the two adapters added onto each end are different)

3. attach fragments to flow cell far away from each other

4. bind the fragment to a nearby primer that can bidn o the adaper

5. amplify dna using pcr

6. dissociate the newly synthesized strand from the original strands

7. repeat until you form multiple clusters

   1. each cluster holds around 1000 identical copies of the original fragments

8. each nucleotide has a color associated to it, but each time DNAP synthesizes a new strand, you watch what color shows to see what gets added → clusters show the color, which is why it is essential to creating the clusters

“sequencing by synthesis”

tldr: each dna fragment is amplified into a cluster to generate a detectable fluorescence signal

single end sequencing

• one side of dna fragment can be read

paired end sequencing

• reads both ends of the same dna fragment

• improve mapping, especially when one ed maps to a unique sequence next to a repeat

  • human genome is mostly repeated due to transposons

next gen sequencing libraries

• contains around 300 bp short DNA fragments

two methods to making libraries:

1. fragmentation of dna by sonication (mechanical sheering), followed by library construction (ligating to add adapters)

2. tagmentation by Tn5 transposase: only one reaction needed to make library because genomic dna is cleaved and tagged with adapters in one go

   • transposase is an enzyme in bacteria, it can insert dna fragment to certain region of the genome

Tn5 and oligo assembly

• Tn5 tranposase recognizes a 19 base sequence, attach this 19 base sequence to our adapter. when the transposome complex comes to cut ~300 bp dna fragment, it tagmented a dna fragment

  • each end of the tagmented dna fragment has an adapter, so it can go through next gen sequencing

secondary, tertiary and quarternary protein structures

held togeter by non covalent interactions like h-bonds, hydrophobic interactions, van der waals and ionic interactions

weak interactions help stabilize protein

a.a. with non polar side chains tend to fold inward while polar side chains fold outwards

polyacrylamide gel electrophoresis (PAGE)

• used for proteins because it has smaller pores

agarose gel electrophoeresis

• has larger pores so mostly used by DNA

• protein are so small they could go right through agarose pores

denaturing sds-page

• sds is a negatively charged detergent

• beta-mercaptoethanol is a reducing agent that can break disulfide bonds

• migration rate in denaturing gel electrophoresis is inversely proportional to the size of the protein

• multi unit proteins usually show up as more than one band on the gel

stains to visualize protein after gel electrophoresis

• coomassie blue staining: binds to the basic and aromatic side chains

• silver nitrate staining: more sensitive, the silver ions from silver nitrate bind to the side chains, followed by reduction of the ion to the free metallic silver. more sensitive than coomassie blue

staining procedure

1. fix the protein in place so it doesnt get washed off

2. incubate gel in solution with dye

3. destain so only where protein is stained remains

immunoblotting (western blotting)

method to visualize protein

1. transfer gel to a membrane

2. use a primary antibody that specifically recognizes our target to bind to our target protein

3. add a second antibody thats coupled with an enzyme for a fluorescent marker

   1. second antibody detects primary antibody, it will bind to the primary antibody which is bound to the target protein, and will light up on oour membrane

IgG structure

• has 2 heavy chains and 2 light chains

• c terminus / tail region is the constant region, reacts with other immune molecules

• n terminus is the variable region, it binds to the target antigen, so second antibody usually binds to the tail region of the primary antibody

protein purification by column chromatography

1. crude lysate (the mixture we want to purify) is applied to the top of the column

2. solvent (buffer) is continuously applied to the top

3. proteins in the lysate interact with the matrix to diff. extents and flow at different rates

4. proteins are collected in different fractions at the other end of the column

3 types of column chromatography

1. ion exchange: separate proteins based on their native charge

2. gel filtration chromatography: separate proteins based on their size

3. affinity chromatography: separate proteins based on their specific binding to the substrate

ion exchange chromatography

retain proteins of similar charge (all protein stuck to bead are same charge)

• column matrix consists of beads carrying either pos or neg charge

• protein bind with bead differently according to their native net charges

• different proteins elute at different salt concentrations

gel-filtration chromatography

retains proteins of similar weight

• column matrix consists of beads containing pores of a defined diameter

• large proteins are excluded from pores and therefore flow through column rapidly

• small proteins enter pores and flow through column slowly

affinity chromatography

specifically bind to selective type of protein therefore more pure product

• specific binding structure is covalently attached to the column matrix beads

• bound proteins can be eluted by increasing ionic strength of washing solution or by competing binding substrate

• relative high levels of purification can be achieved by affinity chromatography

protein purification progression

usually need to do multiple chromatographic steps to successfully purify protein

analyze protein protein interactions by co-immunoprecipitations (co-IP)

1. our protein sample consists of our target protein

2. add antibodies that will bind to our target protein

3. add protein A/G beads that will interact with IgG in high affinity

4. since protein A/G has high affinity towards the antibody (which is bound to the target protein), we can separate our target protein out of solution through centrifugation. keep the pellet

5. then we elute and have our purified complex

6. use a sample buffer to separate target protein from antibody

7. if you perform staining you would be ale to get first impression of how many proteins are interacting or form a complex with your protein of interest

analyzing dna-protein interactions via gel shift analysis

• many protein dna complexes are stable so that they remain together during electrophoresis through native PGE

• this assay figures out if any protein in our cell extract binds to specific dna sequence

1. extract nuclear proteins from cell or tissue and extract dna sequence with 32-P ends

2. protein binds to that dna sequence

3. run the gel electrophoresis, dna with bound protein migrates more slowly than the free probe

4. run autoradiography, which will show the band indicating dna-protein binding complex

doesnt exactly tell you where on the sequence it binds, but confirms that the protein does bind to the sequence

analyzing dna-protein interactions via chromatin IP (ChIP)

determine where in genome a protein is bound in vivo

1. cross-link cells with formaldehyde, dna binding proteins with dna in intact cell

2. isolate the genomic dna and sonicate to shear chromatin

3. short fragments allow determination of precise chromosomal location of chromatin associated proteins

4. add an antibody specific to the protein of interest

5. perform immunoprecipitation to isolate dna bound to the protein of interest, reverse cross link to isolate dna

transcriptome

set of all rna transcripts within cell population

• different cell types have different transcriptomes

• gene expression is tissue and cell type specific

history of gene expression analysis

oldest to newest: northern blot → reverse transcriptase (RT)-PCR → real time qRT-PCR → microarray → bulk RNA sequencing → single cell RNA sequencing

increasing sensitivity and quantitative accuracy

rna extraction by TRIzol and chloroform

cell and tissues with trizol and chloroform under acidic conditions with centrifugation can help separate the different phases

• rna has OH group on 2’ , which contributes to their distinct properties

• aqueous phase: rna

• interphase: dna

• organic phase: proteins and lipids

northern blot

• detects specific rna using complementary labeled probe

• low sensitivity, limited quantitative accuracy

• less commonly used for routine expression analysis

steps:

1. extract rna from organism, electrophoresis to separate rna by size

2. northern blotting: transfer rna to membrane

3. hybridize a labeled probe to the rna of interest and detect probe signal to estimate rna size and abundance

RT-PCR (reverse transcription PCR)

• use dT oligo primer to bind to mRNA poly A tail and allow DNAP to begin polymerizing cDNA strand

• make multiple copies of cDNA

• RT-PCR can detect RNA expression, but it is not reliably quantitative

• useful for cloning cDNA

three RT primer options:

1. random hexamers

2. oligo (dT) primers

3. region specific primers

real time quantitative rt-pcr (RT-qPCR)

• thermal cycler measure fluorescence during each pcr cycle

• widely used but also detects non-specific double stranded dna

• Ct = cycle at which fluorescence crosses a set threshold (during exponential amplification)

• the threshold is placed in the exponential amplification phase

• a lower Ct generally indicates more starting template because it takes less cycles to be detected

Microarray

• array of known sequence dna probes fixed to a solid surface

• thousands of immobilized dna probes are attached to a slide, allowing expression of many genes to be measured at once

  • each spot contains many copies of dna probe designed to recognize a particular transcript

• microarrays measure rna abundance by hybridizing labeled samples to cDNA probes

genome wide method used less often for gene expression profiling today

steps:

1. extract rna from cells you want to determine and convert them to cDNA using reverse transcriptase

2. label the dna with fluorescent probes

3. add them to the microarray and see which spot fluorescences

4. the known oligo sequence that is already in the spot contains our gene of interest

goal is to figure out which genes are present in which cells

cons of microarray

• cross hybridization between similar transcripts and incorrectly annotated probes

• gene expression is measured indirectly through hybridization

• need another independent method (i.e. RT-qPCR) to confirm differential-expression findings

RNA-sequencing

• uses high throughput sequencing to identify and quantify rna molecules, typically after conversion to cDNA

• can reveal transcript boundaries and splicing patterns

• measures changes in transcript abundance across all cell types, development stages or experimental conditions

rna sequencing work-flow

1. extract rna

2. poly (A) selection or rRNA depletion → use RT to make cDNA → build RNA-sequencing library

3. perform next generation sequencing

why is rRNA often depleted before preparing rna-sequencing library

• rRNA makes up most of cellular RNA

• without poly (a) selection or rRNA depletion, many reads would come from rRNA instead of transcripts of interest

rRNA depletion

rRNA binds to DNA probes made to target rRNA, use RNase H to cleave rRNA and bound to DNA, DNA probes are then digested with enzyme

poly A selection

use oligo dT magnetic beads to pick out mRNA with poly A tails

RNA-sequencing libraries (illumina)

two methods:

1. rna fragmentation, cDNA synthesis and library preparation

2. tn-5 mediated tagmentation

profiling gene expression by RNA-seq

rna sequencing estimates gene expression by counting sequencing reads that map to each gene or transcript

access reproducibility

biological replicates: assesses variation among independently collected samples

technical replicates: variability during measurements or processing

validation of rna seq data

• potential errors include incorrect read mapping, ambiguous alignment to repetitive regions, sequencing errors, and biological variation

• assess consistency across independent biological replicates

omics

large scale analysis of biological molecules

• study large sets of biological molecules, different methods describe different layers of cell biology therefore the biology question determines which assay to use

bulk rna seq vs single cell rna seq

• bulk rna sequencing averages rna signals across many cells, and lots of details can be missed due to it being an average

• single cell rna sequencing profiles rna derived molecules from individual cells

  • allows cell types and their states to be distinguished within a heterogenous sample

single-cell rna sequencing workflow

1. isolate cells and capture RNA

2. reverse transcribe RNA and add cell barcodes to help identify cell

3. prepare and sequence the cDNA library

4. assign reads to cell and quantify transcripts and compare expression proteins

   1. use cell barcodes to assign reads

droplet based rna sequencing

each bead carries primers with a distinct cell barcode, a cell barcode identifies the cell of origin of each cDNA molecule (aka, which cell did this cDNA come from?)

1. beads and cell combine into an oil droplet. the all rna from the cell in one droplet get coated with the barcode from the bead its sharing the droplet with

2. reverse transcribe to get barcoded cDNA, then break the emulsion of each droplet so they’re all in the same solution

3. amplify the cDNA and construct the library

4. sequence, the barcode helps identify which cell the cDNA originated from

applications of single cell rna sequencing

identify cell types and states, including rare populations.

study gene expression changes during differentiation

challenges

• each cell contributes little RNA, so data can be sparse

• low abundance transcripts are harder to detect

• a zero count does not always mean a gene is inactive

epigenetics

• gene regulation that can persist without a change in the dna sequence

epigenomics

genome wide analysis of epigenetic features

• measurements by themselves do not establish that a feature is inherited or causes a change in expression

epigenetic memory and cell identity

cell states can persist through cell divisions

• allowing daughter cells to maintain their identity and gene-expression patterns without changing the underlying DNA sequence

chromatin mechanisms of gene regulation

dna methylation, non-coding rna, histone modifications

chromatin immunoprecipitation sequencing (ChIP-seq)

• enrich/selected dna is sequenced and aligned to a reference genome

• regions with more read enrichment indicate target associated chromatin

steps:

1. cross link protein to dna

2. isolate and fragment chromatin

3. bind an antibody to the protein of interest

4. enrich antibody bound chromatin

5. reverse cross-links and purify the dna

   1. dna becomes a sequencing library

6. analyze by next gen sequencing (sequencing and aligning to reference genome)

CUT&Tag

stands for cleavage under targets and tagmentation

• antibody tethers pA/G-Tn5 to a chromatin target

• Tn5 cuts DNA and inserts sequencing adapter tags

  • the fragment that was tagged is amplified by PCR and purified for next gen sequencing analysis

• low input and low background single cell profiling is possible

ChIP-Sequencing vs CUT&Tag

ChIP-Seq:

• targets pull down protein-associated dna

• fragment first, then add adapters

• more input and background

CUT&Tag:

• tether Tn5 with an antibody

• fragment and add adapter tags in sity

• less input and lower background

both rely on antibody specificity and controls

dna methylation

in mammals, mostly happens at CpG sites

dna methyltransferase transfers CH3 onto nucleotide

• maintenance methylation can copy patterns after dna replication

  • during dna replication, methyl can be added to the new strand, can be heritable modification through cell division

bisulfite sequencing

• bilsulfite converts unmethylated cytosine to uracil, but methylated cytosine do not get converted

• during PCR, U templates A. later, DNA copies contain T at the original unmethylated C

• compare reads of the new strand with the original dna sequence to see where there are methylated and unmethylated cytosines

why is an unmethylated c read as t after bisulfate treatment, pcr and dna sequencing

unmethylated c gets converted to u, u complements a which complements t during PCR

cell type specific chromatin accessibility

• less accessible chromatin is mostly associated with repression

• accessible chromatin often includes promoters and enhancers

ATAC-seq: chromatin accessibility

• detect genome wide profile of accessible chromatin

steps:

1. extract chromatin without disturbing its current structure

2. Tn5 inserts adapters into accessible DNA because it cannot reach closed off chromatin

3. tagmentation cuts dna and adds the adapter tags

4. sequence the tagged dna to map the accessible regions

accessible regions tend to include promoters and enhancers, can detect site of gene regulatory element

Hi-C: genome wide chromatin contacts

helps understand how genome interacts with each other

1. fix the chromatin in its current state and cross link

2. digest the chromatin with restriction enzymes, they lose their backbone but because of the crosslinks they are still held together

3. religate the chromatin, now the chromatin bond directly to each other because they were originally close to each other

4. sequence records dna proximity: read pair where one half maps at one position and the other half maps to another position. now you know those spots are touching in 3D

Hi-C heatmap

axes show genomic positions, usually comparing the same chromosome and their base pairs

• intensity of the color show the interaction frequency between two regions, usually a darker color implies those two regions are heavily interacting

why do we study gene functions in vertebrate cells

1. better understanding of genetic diseases of humans and animals

2. some biological processes only appear in vertebrate cells (post translational modifications)

3. protein of interest may need additional accessory factors to function

primary cells

• obtained directly from tissues

• have a limited life span, many cells in adult tissues are quiescent

• they don’t proliferate, or sometimes they do divide but only a finite number of times due to telomere loss

cell lines

• cells maintained through repeated passage; not all are immortal

• can establish cell population to grow

• immortalized cell lines: long term proliferation, sometimes following transformation with viral oncogenes or chemical treatment to force proliferation

• stem cell lines: embryonic stem cells, induced pluripotent stem cells