Term 1 Flashcards
applications of biotechnology
AB production, Ab production, transgenic animals, gene therapy, vaccines, protein pharmaceuticals
do animal growth hormones have therapeutic value in humans
no
what happened when cadaver isolated growth hormones were used? What was done?
transfer of diseases such as creutzfeldt jacob disease and a shortage in availability. In 1980s, human growth hormone was produced using bacterial cells
pharmacists’ role in biotech
1) product evaluation and selection 2) patient education and counselling 3)provision of drug info 4) assistance in patient monitoring 5) drug control and prep
what plant cells have that animals don’t
cell wall, chloroplasts, and centriole
what has DNA/nucleus
chloroplasts, mitochondria and and nucleus all have DNA and nucleus (all double membraned)
how many nucleotides are in the human and mouse genome?
3 x 10^9
A pairs with
T (2 bonds)
G pairs with
C (3 bonds)
do introns or exons code for protein
exons
do higher organisms have more junk DNA (non coding, or “intron” regions)?
yes (only about 1.5% codes for proteins)
mRNA
duplication of genetic material
tRNA
translation of genetic material
rRNA
site for protein synthesis on ribosomes
what differs in RNA in base paits
U instead of T
nucleotide contains
sugar, phosphate, base
where does protein synthesis occur
cytoplasm
what enzyme initiates transcription
RNA polymerase
start codon
AUG
order of translation
initiation, elongation, termination
stop codons
UAA, UAG, UGA (has to be U and then at least one A)
there is at least one unique tRNA for each of the ___ (#) AAs
20
how to sequence DNA (what must we start with)
primer**, need before enzymes work
what kind of DNA do bacteria have
both plasmid and genomic
why manipulate plasmids?
easy to grow and maintain bacterial cells, plasmid DNA is easy to isolate
3 important sections of plasmid
promoter, reporter gene, multiple cloning site
2 key enzymes that made it possible to manipulate sequences of plasmids
restriction endonucleases (cut DNA by recognizing specific BPs), and DNA ligases (link DNA fragments)
what was the first biopharmaceutical
insulin
phases of bacterial growth
lag, log, stationary, death
explain lag phase
number of bacteria does not change with time
explain log phase
number of bacteria increases exponentially
explain stationary phase
no net change in number of bacteria- grow and divide at same rate and die. Happens when overcrowded and nutrients are depleting)Most important biological products (especially secondary metabolites like ABs) are produced here
explain death phase
bacteria decrease with time
any protein can be produced using genetically engineered organisms, but not every type of protein can be produced by any cell type
true
bacteria are not capable of post translational modifications
true- cannot glycosylate, etc
limitations to using bacterial expression systems
no post translational modification (mammalian can), proteolytic cleavage can degrade product, must break/lyse cell to get protein out (are secreted into media in mammalian cells)
animal cells have limits to dividing. How do you overcome this
hybridomas (genetically engineered animal cells with no limits on dividing- this is what cancer cells produce)
other name for bioreactor
fermentor (type where biocatalyst is a living cell)
step one in production of biopharms (synthesis)
cell culture started in small bottles, then small bioreactor, eventually large
types of cultivation systems
-free in suspension, -attached to microspheres or entrapped in matrices that are usually solidified agar, -or immobilized state as monolayers)
types of bioreactors
liquid (aka submerged) (most-saves space and more ammmenable to design, cost effective, easier to maintain, don’t have to worry about monolayers or microspheres are free in suspension)) or solid state (surface)
monitoring of bioreactors
temperature, sufficient substrate (usually carbon source), sugars/proteins.lipids, water, salts, vitamins, oxygen, optimum pH, product and by product removal
types of submerged (liquid) bioreactor operation types
batch, continuous, fed-batch
forms of biocatalyst for submerged (liquid) bioreactors
free cell (enzyme), immobilized cell (enzyme) (packed bed, membrane reactor)
bioreactor systems
stirred tank, airlift, microcarrier (ie fixed bed reactors)
stirred tank bioreactors
-air enters at bottoms, baffles range from 4-8, key factors controlled are pH/temp/oxygen, mixing method is mechanical agitation, requires energy input, bubbles are reduces by an agitator, exhaust gas flows out top
baffle purpose
used to reduce vortexing
what is mixing necessary for
maintain homogeneity, attain rapid dispersion and mixing of components injected into the fermentor, enhance heat transfer and temp control, enhance mass transfer
airlift bioreactors
reaction medium kept mixed and gassed by air or another gas at the base of reactor, seperated into gassed and ungassed regions generating a vertically circulating glow
advantages of airlift bioreactor
simple, no moving parts, less maintenance, less risk of defects and easier sterilization, lower shear rate, both plant and bacterial cells, well controlled flow and efficient mixing, large volume tanks possible which increases the output, enhances oxygen solubility achieved in large tanks with greater pressures, greater heat removal
disadvantages of airlift bioreactor
higher initial investment, greater air and higher pressures needed, impossible to maintain consistent levels of substrate, nutrients and oxygen with circulation and changing conditions, inefficient gas and liquid separation when foaming occurs
micro carrier bioreactors
can have extremely high productivity within a compact size, used for culture of immobilized mammalian cells, us porous glass beads to give large SA,
batch operations
everything put in vessel together, let it grow, watch for products, harvest. Once started, no inlet or outlet unless aerobic system and gas inlet and outlet should be there. Between batches, down time to harvest, empty, clean, sterilize and refill- lowers efficiency)
continuous operation
don’t add substrate all at once, remove throughout. Continuous medium flow- incoming stream (feed) contains substrate and leaving stream (effluent) contains product. Good to maintain bacteria in log phase (keep there longer), contamination more likely
2 assumptions with continuous operations
mixing is vigorous (concentrations are uniform throughout the reactor), effluent concentration is same with reactor contents (chemostat=continuous configurations for cultivation of microorganisms) (conditions don’t change through time=steady state-no accumulation of components ie too much feed, and no depletion of source ie too much effluent)
advantages of batch
-most commonly used because reduced contamination or mutation (due to relatively brief growing period), lower investment, more flexible, higher raw material conversion, easy to operate
disadvantages of batch
lower productivity (maintenance) (down time), increased focus on instrumentation, greater expense in preparing several subcultures for inoculation, higher labor and process control costs, batch to batch variability, accumulation of inhibitory products
continuous reactors advantages
increased opportunities for system investigation and analysis, higher degree of control, results are more reliable and reproducible (quality), efficient, higher productivity, uniform product, no accumulation of inhibitory proteins
disadvantage of continuous reactors
higher contamination and cell mutation risk (long growing periods and continuous reactions, higher investment, less flexible
fed batch
modification of batch cultivation in which nutrient is added intermittently
cell bank (and 2 types)
when cell line is to be used over many cycles- master cell (expanded to WCB) and working bank (ones currently use and experiment with)
cell banks must be tested for
contaminents, and some for viruses
higher speed centrifuge is required for separation of smaller particles
true
inclusion bodies
bacterial proteins aggregate - often occur when bacterial cells overproduce proteins through the use of plasmid expression
advantages and disadvantages of inclusion bodies
A- relatively simple to recover, protected from proteolytic cleavage D- biologically inactive- must be denatured first to be solubilized then refolded for activity, inaccurate assessment of recovery-can’t directly measure, recovery of proteins requires cell breakage/protein sedimentation/pellet washing.
disrupting cells can be 2 ways
-mechanical (sonication and liquid shear homogenization) and non (autolysis, osmotic shock)
if proteolytic degradation will occur, purification should be carried out at ___degrees celsius
4
inclusion bodies dissolve in denaturing agents such as
SDS, urea, guanidine HCl
if molecules are interlinked by disulfide bonds, must add ___ agents such as
reducing- mercaptoethanol, dithiothreitol (DTT)- they may enhance or facilitate the solubilization of protein inclusion bodies
presence of glycosylation can affect
solubility, stability, serum half life, pharmacological fx, immunogenicity
protein glycosylation is directly determined by DNA sequence
false
why can’t most proteins be sterilized by the standard methods
denaturation at high temperatures
components of parenteral formulations
solubility enhancers, anti adsorption and anti aggregation agents, buffer, antioxidants, preservatives, osmotic agents, carrier systems, active ingredient
why are solubility enhancers needed in parenteral formulations
generally a high concentration is used in biotech drugs, and this can cause proteins to aggregate and precipitate out (lowers potency, increasing change of immunogenicity). Interaction with excipients instead of other proteins= MOA
when does aggregation of proteins occur
hydrophobic or electrostatic interactions between different proteins
approaches to enhance solubility
proper pH and ionic conditions, additions of AA like lysine and arginine (tend to solubilize t-PA), adding surfactants (SDS), sugars like glucose and sucrose
what is used to prevent insulin adsorption
albumin- has higher affinity for surfaces (competition)
what is likely to form fibrillar precipitates
(long rod shaped structures); low concentrations of phopholipids and sufactants, insulin; solve by selecting proper pH often
common buffer system components in biotech
phosphate, citrate, acetate
when freezing conditions (lyophilizing)- buffers can ___ or form _____ and pH changes will be minimized
crystalize (Na2 faster than Na), or don’t crystalize- form amorphous systmes
what is most often oxidized
AA
how to add an antioxidant
replacement of O2 by inert gases helps reduce oxidative stress, special airtight vials, vitamins A,C,E, AAs, chemicals (selenium, acetic acid, sodium citrate)
preservatives- what to take into consideration
lowest concentrations possible to minimize SE, want as not toxic and immunogenic as possible
preservatives-bacterio static or cidal
ususally static- stop growing not kill
what do osmotic agents do
modulate tonicity; can use preferential exclusion to stabilize proteins (osmotic agent increases interaction of protein with solvent so that it is in the solubilized state not precipitating out) (they themselves are excluded from protein surface layer) (puts protein in solubilized state longer)
problem with strong preferental exclusion
enhances tendency of proteins to self associate (add osmotic agents to enhance interaction of solvent with protein and they themselves are excluded from protein surface layer)
what is product purity
greater than or equal to 99%
using serum may do what
introduce contaminating proteins (especially fetal calf serum-often used in mammalian cell cultures), can have viruses/bacteria/prions/fungi, even when sterile may contain endotoxin (should be eliminated during purification)
contaminants present in recombinant protein products from bacterial and non sources are 1 of 3 things
host related, product related, or process related
most frequent source of virus introduction- how to solve
animal serum- can also introduce bacteria, mycoplasmas, fungi and endotoxins- move towards defined growth media where serum levels are significantly reduced
how are viruses inactivated (physical and chemical) or removed
heat, irradiation, sonication, extreme pH, detergents, solvents, certain disinfectants. Removed= chromatography, filtration, precipitation
how to remove bacteria from product
size allows simple sterile filtration
how to remove pyrogens
not by sterile filtration, usually at least one step of ion exchange chromatography (anionic exchange) to remove the negatively charged pyrogens
what are pyrogens and what do they cause
usually endotoxins of gram negative bacteria, causes strong fever and can be fatal
methods to detect if cellular DNA is present in our final product (we want it removed)
radiolabelled nucleotides, dye binding
why are protein contaminants a problem
may be recognized as antigens by patient and on repeated use may show an immune reaction- can be misinterpreted as the protein of interest we want to cause benefit causing this immunogenicity
what are sources of protein contaminants
growth medium, host proteins of cells (which are similar in structure and might be co purified with desired product), ligands from affinity columns used in the purification process
serum free media is favored for large scale manufacturing for pharmaceutical proteins
true
barriers to extensive use of serum free media
insufficient cell growth, productivity, cells grow slower
what is often the rate limiting step in development of a biotech pharmaceutical
chromatography- usually 2-3 different chrom procedures will be used in succession to isolate the protein of interest
phases of chromatography
stationary (insoluble matrix), mobile phase (solution passed through or over stat phase), separation based on differences between two phases- goal is to elute proteins off column one at a time
direct immunofluorescence
add fluorescent body to antigen- greater binding will give greater fluorescence. Can check for type and quantity of protein. Very expensive
indirect immunofluorescence
attach antigen but use unlabled antibody not fluorescence tagged. Then use common antibody against all immunoglobulins (ie all antibodies) with fluorescence tag
difference between immunofluorescence and ELISA
immuno detects fluorescence, ELISA detects substrate
inderect ELISA
give antibody, binds antigen, add antibody linked to enzyme, substrate converted to product- more enzyme=more color=more antigen
steps in ensuring biotech quality
plasmids and host cells, protein stability, process validation (for purification), final product batch check
stability testing of final labeled products include
freezing, heating, denaturing
-long term integrity and sterility are also monitored
it is important to generate proteins with an N terminus (amino) like found in authentic protein because- (and what is this called)
if not, final product may be a mix of several methionyl variants or contain proteins lacking one or more residues from the amino terminus (called amino terminal heterogeneity). NOT desirable because it causes difficulties in purification and characterization of proteins
in what direction is DNA extended for PCR, and what does it first require
5’ to 3’, needs primer and taq polymerase accomplishes add on
3 steps of PCR
melt, anneal (cool), extend (DNA synthesis)
things required for PCR optimization
buffers (KCL and tris usually-can contain others), MgCl2, primer design, cycle number
what is the ideal length to sequence for PCR and why, and the ideal temperature and GC content, ideal primer length, acceptable number of runs and repeats
18-30 nucleotides, 50-70 celsius, 40-60% GC, less than 5 hairpins. Too short=low specificity, too long= decrease template binding efficacy (higher probability of hairpins, etc), ideal primer length 18-24, 4 bp=acceptable run and repeats (too long increases mis priming and non specific annealing)
why is magnesium needed for pCR
acts as a cofactor for taq polymerase (needs it to fx), binds DNA and affects primer template interaction (affects interaction with taq too), too much leads to non specific/less stringent binding, too little is a reduced yield
what are hairpins
formed by intramolecular interaction
what are self dimers (homodimers)
intermolecular interactions between the two same primers
cross dimers (heterodimers)
intermolecular interactions between the sense and antisense primers
how much lower should the annealing temperature be than the melting temperature
5 degrees celsius
what is the half life of taq at 95 degrees Celsius and why does this matter
-30 minutes: more than 30 cycles of denaturation times of 1 minute, taq is not very effective- therefore ideal cycle number is 25-40. After 30 cycles, won’t see as much yield (plateau effect)
the Tm for PCR should not be more than
60 degrees
how should primers in PCR end
3’ in G or C or CG or GC- prevents breathing of ends and increases efficacy of priming
what is site directed mutagenesis and what does it need
creates a mutation at a defined site- requires a known template sequence; a mismatched oligonucleotide is extended, incorporated into a strand of DNA that can be cloned
what is ASO
antisense oligonucleotide- short DNA analogue that hybridizes with complimentary mRNA in a sequence specific manner (can result in inhibition of gene expression giving reduced levels of translation of the target transcript)- inhibits ribosomes so they can’t bind and you can’t get expression
what are ASOs being used for?
researched as potential drugs for cancer and other diseases (“virsen, mersen”) especially linked to dysregulated gene expression
what does an effective ASO need to be designed at
regions where mRNA is accessible for hybridizations (usually terminal end, internal loops, hairpins, joint sequences, bulges or 10 or more
how to deliver ASO (the problem)
-unmodified and naked has a net negative charge that can barely penetrate the plasma membrane. Fix with electroporation (by electric field), microinjection, vectors (cationic lipid carriers), covalent conjugation of ASO to a macromolecule (like dendrimer and cell penetrating peptide- CPP4)
what does it take to bring a drug to marker?
about 1 billion, and 5-10 years
what was the first genome sequenced
bacteria
how many bps does the human genome have
3.2 billion (includes all DNA on all chromosomes and genes, intergenic sequences and repeats)
eukaryotes can have 5-6 genomes
false- 2-3 (nuclear, mitochondrial, plastid)
what makes something druggable
if the drug can bind to that target. May or may not involve disease therefore separate into non disease and gene disease
what are me too drugs
another drug already doing jib, new drug works too
what is the reason for genetic differences in people
genetic variation- 2 kinds 1= most common=SNPs- single nucleotide polymorphisms (single base pair positions which different alleles exist), 2= insertion or deltion of one or more nucleotides
polymorphism
genetic variation observed at a frequency of more than 1% in a population
SNPs- usually have no phenotypic effect
true- very common, lots in uncoding regions, some are alleles of genes
tandem repeat polymorphisms
common polymorphism, variable length sequences that are repated in tandem (microsattelites are 1-6 units, mini are 14-100)
pharmacogenetics vs pharmacogenomics
genetics= variability in drug response determined by single genes, genomics is multiple genes
how many bacterial genomes are completely sequenced
over 5000
targets in bacteria must be two things** main, and a third
selective (only present in bacteria not host-potentially cell wall, inhibiting unique enzymes, disrupting bacterial protein synthesis), essential (essential for bacterium so when we knock it out it dies), wide spectrum (present in all subtypes we target)
c value paradox
complexity doesn’t correlate with genome size
N value paradox
complexity doesn’t correlate with genome number
k value paradox
complexity doesn’t correspond with chromosome number
if you have increased number of cells you have increased number of
genes
karyotyping
gene matching in which you compare chromosomes
why is the rate/mouse model of choice for pre clinical drug discoveries?
mouse and human synteny; humans chromosomes can be cut into about 150 pieces and shuffled to reasonable approximation of mouse
why do comparative genomics
tells us what are common and unique between species at genome level, genome comparison is the surest and most reliable way to ID genes and predict fxs and interactions, fxs of human genes and other DNA regions can be revealed by studying their counterparts in lower organisms
how many bp in the human genome
3 billion
how many human genes are shared with primates? fruit fly?
over 98%, about 60%
what is the CGAP and what have scientists learned using it
Cancer gene anatomy project, in cancer cells certain genes are damaged or switched off while others are on
what are the properties of a database
structured, searchable, updateable, cross linked, publicly available
2 types of databases at NCBI
1) primary- data is submitted by the person who generated it (original submissions by experimentalists, content controlled by submitter) 2) derivative databases- built from primary data (supplement info and create new database)
gene annotation and the 2 steps
process of attaching biological information to sequences in databases. 2 steps: identify genes on genome, attach biological information to genes and genome.
forms of annotations in databases
genome annotation, variation mapping, comparative genomics, functional genomics
alignment concept (and what is optimal)
mutual arrangement of 2 sequences that exhibits where they are similar and where they differ- an optimal alignment is with most correspondences and least differences
2 bases for alignment
1) structural 2) evolutionary
structural alignment
when 2 protein sequences have more than 20-30% identical residues aligned, the 3D structure is very similar, and form often follows function (so it implies similar fx as well). Sequence alignment is often an approximate predictor of underlying 3D structural alignment
evolutionary alignment
similarity is an observable quantity that may be expressed as % identity. Enables to determine if two sequences display sufficient similarity. Evolutionary related
what is drugbank
a database on drugs, targets and enzymes
4 types of chromosome mutation
duplication, deletion, translocation, pericentric inversion
what does germ ilne gene therapy aim at
the introduction of genes into germ cells of omnipotent embryonal cells, human germ cell is not ethical
what is somato gene therapy
the introduction of a gene into somatic cells
what is ex vivo gene therapy
cells removed from patient, cultured ex vivo, can be provided with therapeutic gene, the infused or reimplanted back into patient (usually with retroviral vectors)
what is in vivo gene therapy
organs like lung, brain, heart, etc are not suited for ex vivo, so somatic gene therapy can only be performed by administering gene of interest either locally or systemically
where is somatic gene therapy aimed
at cells or organ where the disease is manifested
how is stable gene transfer accomplished (how does the therapeutic gene integrate into host cell chromosomal DNA)
retrovirus or adenovirus mediated gene transfer, or at low efficacy after non viral mediated DNA transfer
what is therapeutic gene transmitted to
progeny cells
what does episomal transfection with and without and organ cause
with- stable gene transfer and transmitance to progeny cells
without- only transient infection (episomal DNA is lost upon division)
what is the most common approach employed for cancer gene therapy
cytokine genes like interferon, interleukins, and GM CSF- they induce a local inflam reaction which destroys both tumor and metastases
what is a suicide gene- give an example
another way to approach cancer gene therapy, mediate direct cytotoxic or antiproliferative effects on tumor cells and are only effective for localized tumors- phosphorylated ganciclovir is incorportated into DNA of dividing cells, leading to elimination of DNA chain elongation, resulting in cell death
what does the bystander effect occur through
gap junctions to neighboring cells
tumor suppressor genes such as __ are often targeted in cancer, or ___ genes are targeted at oncogenes
p53 (which are mutated), antisense are targeted at oncogenes to reduce or abolish their expression. These are for direct cytotoxic or antiproliferative effects and only effect localized tumors
a way to target cancer is to protect _____ from toxic effects of chemotherapy by _____
hematopoietic stem cells (HSCs) from the toxic effect of chemotherapy by inserting a gene that confers drug resistance (like MDR-1 ie multidrug resistance gene)
what does MDR-1 allow patients to do
multidrug resistance gene protects hematopoietic stem cells from toxic chemotherapy, so patients are expected to tolerate higher doses of chemotherapy and thus increase effectiveness
viral vs non viral gene transfer
viral is more efficient, non is naked DNA inserted in liposome and is not as efficient but has no known safety issues
methods of non viral gene transfer
injection of naked DNA, particle bombardment (gene gun), entrapping DNA into liposomes
injection of naked plasmid DNA for non viral gene transfer
simplest, reasonable efficacy in muscle and skin, requires a lot of DNA because not that efficient, easy, safe, suitable for large gene constructs, can also be introduced with gene gun (microscopic gold or tungsten pieces forced with it)
how liposome encapsulated DNA transfer non viral works; methods to help it
DNA is negatively charged so put in positively charged liposome; fuse with membrane of target cell, DNA released into cell. Problem is few that are released into cytoplasm are expressed- fix with adding peptides or virus shells to complex to minimize breakdown of DNA, or attach antibodies for targeting purposes. Safe, efficiencies low
how have DNA liposomes and non viral gene transfer been used for CF?
CFTR =cystic fibrosis transmembrane conductance regulator gene that codes for protein that transports chloride (CF have defect in this),clinical trials on DNA/liposomes instilled in patients’ noses (same secretory defect as lungs and easier to harvest and measure cells)
gene transfer using recombinant viruses (viral vectors) is good because
virus has natural capacity to infect cells and deliver genes to nucleus, much more efficient than non viral. Virus is inactivated so can’t reproduce (retro, adeno, adeno associated)
if viruses are used to transport genes, must:
be replication defective, no undesirable properties, able to accommodate therapeutic gene in its viral genome
ideal vector for gene transfer
high efficiency (many cells infected or transduced), convenience and reproducability of production, ability to transduce dividing and non dividing cells, ability to integrate into a site specific location in host chromosome or successfully maintained as stable episome, transcriptional unit can be controlled by its regulatory elements, ability to target desired type of cell, no immune response
what is a retrovirus and how does it work
genome has two copies of single stranded RNA; goes through reverse transcription using RNA as template. Must adhere and enter cell. Uses integrase to insert its reverse transcribed double stranded DNA into human genome and may result in mutagenesis (forms pro virus). Proviral DNA is transcribed and translated into viral proteins which bud from cell wall
retrovirus vector properties that make it suitable for gene transfer
affect a wide variety of cells with high efficiency, proviral copy stably integrate into chromosomal DNA of cell giving life long correction of target cell type and descendants, sequences for replication can be separated into cis and trans which enables generation of replication defective recombinant retroviruses
recombinant retrovirus systems consist of two building blocks
retroviral vector (to transfer gene-ie piece of RNA with gene), retrovirus packaging cell (only needed to produce replication defective virus ie the vector)
what are retroviruses used predominantly in
ex vivo gene transfer; ususally need growth factors to stimulate division of target cells and increase transfer efficiency
what was the first gene therapy protocol for
adenosine deaminase (ADA) deficiency (lethal inherited disorder leading to severe combined immunodeficiency) (destroys T and B cells because can’t produce uric acid)
what are gene marking studies
have no therapeutic intent- aim at demonstrating that an exogenous gene can be safely transferred to patient and determine how long this gene is detectable in the patient’s target cells
problems with gene therapy
short lived (rapidly dividing nature of cells prevents long term correction-need multiple rounds of therapy), immune response, viral vectors (toxic or inflam response or cause disease), multigene disorders (hard to treat- ie heart, BP, alzheimers, arthritis, DB), may induce a tumor (insertion based mutagenesis)
how can genetic material of animals be manipulated
insertion (transgenes), replacement, deleted (knockout)
gene therapy vs transgenic
gene therapy- needs a vector and attempts to replace or repair in a developed animal, vs transgenic is grown from a genetically modified embryo from birth
what is the most common way to produce transgenic animals
DNA microinjection (inject into larger, male pronucleus, prior to first cell division so that all cells will contain the transgene, can be passed on 20-25% of time in detectable levels to offspring)
most efficient way to produce transgenic animals
in vitro by homologous recombination in embryonic stem cells; derived from mouse blastocyst (can have DNA modified while retaining ability to contribute to somatic and germ cell lines), done by microinjection or transfection then implant into blastocyst and transfer to surrogate animal
what happens when you use hematopoietic stem cells vs ES cells
ES cells change the whole germ line, hem repopulate a specific somatic cell line(s) (more similar to gene therapy)
animals can serve as bioreactors to synthesize recoverable quantities of therapeutically useful proteins, especially in milk (harvested easily mechanically or manually by milking)
true
gene pharming
recombinant genes for desired protein are fused to requlatory sequences of animals’ milk producing genes (targetted exclusively to mammary tissue) (animals not endangered at all)
how are yields and price of gene pharming different vs recombinant cell culture
they are 10-100x higher, price is 75% less expensive, and can undergo post translational modification
advantages of gene pharming
protein produced in large quantities in milk, can be harvested mechanically or manually simply by milking, yeileds are 10-100 greater, 75% less expensive, can have post translational modification if required
what are knockout mice
endogenous gene has been inactivated by replacing it with a null allele (good tool to examine gene function in vivo)
what is genetic ablation
aka cell ablation or genetic amputation, used to suppress growth of a specified cell line or cell type in an animal rather than suppress gene expression. transgene is under control of gene promoter that is only active in a certain cell population
