In hybridoma technology, fused cells are grown in HAT medium. Aminopterin in HAT forces cells to use the nucleotide salvage pathway, and the surviving hybridomas owe their survival to

HGPRT contributed by the B-cell partner, with immortality from the myeloma

HGPRT contributed by the myeloma, with immortality from the B cell

resistance to aminopterin coded on a plasmid

their ability to grow without any nucleotides

Microcarriers are used in bioreactors mainly to

provide a large attachment surface so anchorage-dependent cells can be grown in a stirred suspension

supply oxygen directly to the cells

remove carbon dioxide from the medium

Which dye and counting device are used together to measure the viable cell count of an animal cell suspension?

trypan blue, which stains only dead cells, on a haemocytometer

ethidium bromide on an electrophoresis gel

phenol red in a spectrophotometer

Why is CHO (Chinese hamster ovary) the standard host for producing therapeutic monoclonal antibodies rather than E. coli?

CHO performs human-like glycosylation and secretes correctly folded antibodies, which E. coli cannot

CHO grows faster and cheaper than E. coli

E. coli cannot replicate plasmids

A conditional knock-out using the Cre–lox system allows a gene essential for embryonic development to be studied in the adult because

Cre recombinase, expressed only in a chosen tissue or time, deletes the loxP-flanked gene only there and then

the gene is deleted in every cell from the zygote onward

loxP sites increase the gene’s expression

Cre protects the gene from mutation

Which of the following are correctly paired product and type? Select all that apply.

Penicillin — secondary metabolite (non-growth-associated)

Ethanol — primary metabolite (growth-associated)

Polyhydroxybutyrate — a microbial storage bioplastic

Penicillin — secondary metabolite (non-growth-associated)

Ethanol — primary metabolite (growth-associated)

Polyhydroxybutyrate — a microbial storage bioplastic

Citric acid — an antibiotic from Streptomyces

When recombinant human insulin is expressed in E. coli, a common downstream challenge is that the protein

accumulates in insoluble inclusion bodies that must be solubilised and refolded

is hyper-glycosylated and must be deglycosylated

is secreted into milk and must be separated from casein

integrates into the host chromosome and cannot be recovered

Mouse monoclonal antibodies are "humanised" before clinical use mainly to

reduce the patient’s immune response against the foreign (mouse) protein

make them bind antigen more tightly

allow them to be grown in E. coli

In screening soil for new antibiotic producers, an isolate is often judged a candidate on a primary screen by

a zone of growth inhibition it produces around itself on a lawn of a test organism

Fetal bovine serum is added to many animal cell media chiefly to supply

antibiotics against contamination

Which of the following are features of making a transgenic animal by pronuclear microinjection? Select all that apply.

DNA is injected into a pronucleus of a fertilised egg

The transgene integrates at a random site, often in multiple copies

It can be used to produce a therapeutic protein in milk (gene pharming)

DNA is injected into a pronucleus of a fertilised egg

The transgene integrates at a random site, often in multiple copies

Integration is targeted to a chosen locus by homologous recombination

It can be used to produce a therapeutic protein in milk (gene pharming)

Induced pluripotent stem (iPS) cells are produced by

reprogramming adult somatic cells with factors such as Oct4, Sox2, Klf4 and c-Myc

fusing B cells with myeloma cells

removing the nucleus from an egg

culturing the inner cell mass of a blastocyst

Animal and Microbial Biotechnology — Cell Culture, Hybridoma and Stem Cells, Transgenic Animals, and Industrial Microbes

The second chapter of Section 5 takes the other two sub-headings of the syllabus: Animals and Microbes. Animal cell technology first — culture media and growth conditions, preservation of cells and tissue, anchorage-dependent against suspension culture, the kinetics of animal cell growth, and the micro- and macro-carriers that let anchorage-dependent cells be grown at scale. Then the cells and animals that biotechnology makes from them: hybridoma technology for monoclonal antibodies, stem cell technology, animal cloning, transgenic animals, and knock-out and knock-in animals. Finally microbial biotechnology — the production of biomass and of primary and secondary metabolites (biofuels, bioplastics, industrial enzymes, antibiotics), large-scale production and purification of recombinant proteins, clinical, food and industrial microbiology, and the strategies used to screen for new products.

1. Animal cell culture — media, growth conditions and preservation

Animal cells are more demanding than microbes or plant cells. Standard basal media such as DMEM (Dulbecco’s Modified Eagle Medium) supply glucose, amino acids (including glutamine, a key carbon and nitrogen source), vitamins and inorganic salts, and are usually supplemented with 5–10% fetal bovine serum (FBS) for growth factors, hormones, attachment and carrier proteins, and protease inhibitors. Serum is variable and expensive, so serum-free and chemically defined media are increasingly used, especially for therapeutic production where a defined composition and freedom from animal contaminants matter. The medium holds a CO₂/bicarbonate buffer with phenol red as a pH indicator, and cultures are kept at 37 °C in a humidified 5% CO₂ incubator to maintain about pH 7.2–7.4. Antibiotics such as penicillin–streptomycin are added against contamination, to which animal cultures are very vulnerable because they grow slowly.

A primary culture is made straight from a tissue (often disaggregated with trypsin and EDTA); once subcultured it becomes a cell line. A finite cell line divides a limited number of times and then stops (the Hayflick limit) through replicative senescence, whereas a continuous (immortalised) line — HeLa, CHO, HEK-293 — divides indefinitely after transformation or immortalisation. Preservation is by cryopreservation: cells are frozen slowly (about 1 °C per minute) in medium with a cryoprotectant, usually 5–10% DMSO or glycerol, to keep ice crystals from rupturing the cells, stored in liquid nitrogen at around −196 °C, and thawed rapidly. The cryoprotectant lowers the freezing point and reduces intracellular ice; slow cooling lets water leave the cell osmotically before it freezes inside.

2. Anchorage dependence, growth kinetics, and micro- and macro-carrier culture

Most normal animal cells are anchorage-dependent: they must attach and spread on a surface to grow, and stop dividing when the surface is covered (contact inhibition). They are grown as a monolayer on treated plastic (flasks, roller bottles) and subcultured by detaching them with trypsin. Transformed and many tumour cells are anchorage-independent and grow in suspension, which is far easier to scale; hybridomas and engineered CHO lines are often adapted to suspension for exactly this reason. Growth in a batch follows lag, exponential (log) and plateau phases, and in the exponential phase the population doubling time is td = ln 2/mu, mu = ln(N₂/N₁)/(t₂ − t₁); animal cell doubling times are long, typically around a day. Viability and number are read with a haemocytometer and trypan blue, which stains only dead cells because their membranes have lost integrity.

The problem anchorage dependence creates is surface area: a monolayer offers little area per unit volume, so scaling means more surface, not just more medium. Microcarriers solve this — small (around 100–300 micrometre) beads of dextran, gelatin or plastic, sometimes charged (DEAE) or collagen-coated, suspended and gently stirred in a bioreactor so that anchorage-dependent cells grow on their surface. Because surface area per bead scales with its diameter squared while the number of beads per unit volume scales with the inverse cube of the diameter (1/d³), total area per unit volume scales as 1/d, so a given mass of small beads offers a very large total area: microcarriers turn a surface problem into a stirred-tank problem. Macroporous carriers go further, with pores the cells grow inside, which also shields them from shear and allows very high densities; packed-bed and fluidised-bed reactors use such carriers for perfusion culture. Hollow-fibre reactors grow cells on the outside of semipermeable fibres through which medium flows, reaching tissue-like densities.

🎯 Why small beads give more area

For a fixed total bead mass (fixed total volume), halving the bead diameter multiplies the number of beads by eight (volume scales as d³) while each bead’s area falls only fourfold (area scales as d²), so the total surface area doubles. Smaller carriers therefore pack more growth area into the same tank — the usual GATE reasoning behind a microcarrier numerical.

3. Hybridoma technology and stem cell technology

Hybridoma technology (Köhler and Milstein, 1975) makes a single, defined (monoclonal) antibody. A mouse is immunised with the antigen; its antibody-secreting B cells (plasma cells) — which cannot divide indefinitely — are fused with an immortal myeloma cell line using PEG. The fused cells are grown in HAT medium (hypoxanthine, aminopterin, thymidine) to select hybridomas: aminopterin blocks the de novo nucleotide pathway, so cells must use the salvage pathway, which needs the enzyme HGPRT. The myeloma partner is chosen to be HGPRT-deficient, so unfused myeloma cells die in HAT; unfused B cells die of their finite lifespan; only a B cell × myeloma hybridoma has both the B cell’s HGPRT (surviving HAT) and the myeloma’s immortality (dividing forever). Hybridomas are then screened (by ELISA), the positive ones cloned by limiting dilution, and each clone secretes one antibody. Therapeutic antibodies are humanised or made fully human (by chimaeric constructs, transgenic mice or phage display) to avoid the immune response a mouse antibody provokes.

Stem cells are unspecialised cells that self-renew and can differentiate. They are graded by potency: a totipotent zygote can make the whole organism and the placenta; pluripotent embryonic stem (ES) cells, from the inner cell mass of the blastocyst, can make any cell of the body but not the placenta; multipotent adult stem cells (such as haematopoietic or mesenchymal stem cells) make a limited range within a lineage; and unipotent cells make one type. Induced pluripotent stem (iPS) cells — Takahashi and Yamanaka, 2006 — are adult somatic cells reprogrammed back to pluripotency by forcing a small set of transcription factors (Oct4, Sox2, Klf4, c-Myc), which sidesteps the ethical problems of embryos and gives patient-matched cells. Stem cells are used in regenerative medicine (bone-marrow transplants are the established case), disease modelling and drug testing.

⚠️ Totipotent in plants, pluripotent in ES cells

Plant cells are described as totipotent because a somatic cell regrows a whole plant (Chapter 1). In animals, embryonic stem cells are pluripotent, not totipotent — they make the body but not the placenta — and only the zygote and its earliest blastomeres are truly totipotent. A question using both words is testing this distinction.

4. Animal cloning, transgenic animals, and knock-out and knock-in animals

Animal cloning by somatic cell nuclear transfer (SCNT) takes the nucleus of a somatic (body) cell and injects it into an enucleated egg (an oocyte whose own nucleus has been removed); the reconstructed egg is activated, develops into an embryo and is implanted in a surrogate. The lamb Dolly (1996) was the first mammal cloned from an adult somatic cell, proving that a differentiated nucleus can be reprogrammed by egg cytoplasm to totipotency. The clone’s nuclear genes come from the donor of the nucleus, but its mitochondria come from the recipient egg, so it is not a complete genetic copy. SCNT is used to multiply elite livestock and, as therapeutic cloning, to derive patient-matched ES cells.

Transgenic animals carry a deliberately added foreign gene. The classical route is pronuclear microinjection — DNA is injected into the male pronucleus of a fertilised egg and integrates randomly; others are retroviral or lentiviral infection, and ES-cell-based methods. Uses include gene pharming (producing therapeutic proteins in milk — antithrombin from transgenic goats was the first such product approved), disease models, and faster-growing or disease-resistant livestock. Knock-out and knock-in animals are made by gene targeting: a construct with regions homologous to the target gene is introduced into ES cells, and in a rare cell it integrates by homologous recombination at the right locus. A knock-out disrupts the gene (to find out what it does by removing it); a knock-in replaces it with a modified or reporter version, or inserts a sequence at a defined site. Selected ES cells are injected into a blastocyst to make a chimaera, which is bred to get the change through the germ line. Conditional knock-outs use the Cre–lox system — loxP sites flank the target, and Cre recombinase, expressed only in a chosen tissue or time, deletes it there — so a gene essential for development can still be studied in the adult. CRISPR–Cas9 (Chapter on molecular tools) now makes all of this far faster and does not require ES cells.

⚠️ Random integration versus gene targeting

A “transgenic” animal from pronuclear microinjection has the transgene inserted at a random site, often in several copies. A “knock-out/knock-in” comes from homologous recombination at a defined locus, so position is controlled. A question that stresses homologous arms, a specific locus or Cre–lox is about targeting, not random transgenesis.

5. Microbial biotechnology — biomass and metabolites

Microbes are grown for biomass itself and for metabolites. Primary metabolites are made during growth and are often growth-associated — ethanol and lactic acid (fermentation), amino acids such as glutamate and lysine, organic acids such as citric acid (Aspergillus niger), and vitamins. Secondary metabolites are made typically after growth slows (idiophase) and are not needed for growth — the antibiotics (penicillin from Penicillium, streptomycin from Streptomyces), and many pigments and toxins. Biomass products include single-cell protein and baker’s and brewer’s yeast. The Luedeking–Piret relation separates the two kinds: specific product formation qp = alpha·mu + beta, where a growth-associated product has beta near zero and a non-growth-associated one has alpha near zero.

What microbes make at industrial scale
Product group	Examples	Typical organism
Biofuels	Bioethanol (from sugars or starch), biobutanol, biogas (methane)	Saccharomyces cerevisiae; Clostridium; mixed anaerobes
Bioplastics	Polyhydroxybutyrate (PHB) and other polyhydroxyalkanoates stored as carbon/energy granules	Cupriavidus necator (Ralstonia eutropha)
Industrial enzymes	Amylases, proteases (detergents), cellulases, lipases, rennet	Bacillus species; Aspergillus; engineered hosts
Antibiotics	Penicillin, cephalosporin, streptomycin, tetracycline, erythromycin	Penicillium, Streptomyces, other actinomycetes

Yields and productivity are the engineering language for these processes (Section 4 of the paper develops the kinetics). The biomass yield on substrate is Y_x/s = biomass formed per substrate consumed, and the product yield Y_p/s is product per substrate; volumetric productivity is product (or biomass) made per unit volume per unit time. Worked example: a fermentation consumes 50 g of glucose per litre and forms 20 g of ethanol and 2 g of cells per litre. Then Y_p/s = 20/50 = 0.40 g per g (against the theoretical maximum of 0.51 g ethanol per g glucose, so a fermentation efficiency of 0.40/0.51 = 78%) and Y_x/s = 2/50 = 0.04 g per g. If this takes 10 hours, the ethanol productivity is 20/10 = 2.0 g per litre per hour.

6. Recombinant proteins, applied microbiology and screening for new products

Large-scale production and purification of recombinant proteins and metabolites. A gene is expressed in a host (Section 6 covers the vectors): *E. coli* is cheap and fast but cannot glycosylate and often deposits protein in inclusion bodies that must be solubilised and refolded; yeast (Saccharomyces, Pichia pastoris) secretes and glycosylates and grows to high density; mammalian cells (CHO above all) give human-like glycosylation and are the standard for antibodies and complex glycoproteins. Production runs in batch, fed-batch or perfusion mode in stirred-tank or wave bioreactors, with pH, dissolved oxygen, temperature and feed controlled (the control loops are in Section 4). Downstream processing then recovers the product: cell removal (centrifugation, filtration), release if intracellular (homogenisation or lysis), and purification — the sequence usually running capture, intermediate purification and polishing, with chromatography (ion exchange, affinity such as Protein A for antibodies, size exclusion, hydrophobic interaction) doing the fine separation, ending in formulation and sterile filtration. Insulin was the first recombinant therapeutic protein (approved 1982).

Clinical, food and industrial microbiology. Clinical microbiology identifies pathogens and tests their antibiotic susceptibility and underlies diagnostics and vaccine production. Food microbiology covers both the microbes that spoil or poison food and those that make it: lactic acid bacteria for yoghurt and cheese, yeast for bread and beer, and starter-culture fermentations. Industrial microbiology is the production side above — enzymes, acids, solvents, antibiotics, vitamins — plus bioremediation and biomining.

Screening strategies for new products. New producers are found by primary screening — qualitative tests on many isolates (soil is the classic source of antibiotic-producing actinomycetes), such as a zone of inhibition around a colony on a lawn of a test organism (the crowded-plate and agar-overlay methods) — followed by secondary screening, which quantifies yield, spectrum and novelty in shake flasks and small fermenters. Enrichment culture biases a mixed inoculum toward the organism of interest by choosing the carbon source, nutrients or conditions only it exploits. Once a producer is found, its yield is raised by strain improvement — repeated rounds of mutagenesis and selection, and now metabolic engineering — because a wild isolate makes only a trace of what an industrial strain must. Modern screening adds high-throughput robotics and metagenomic libraries that reach the many microbes that cannot yet be cultured.

Key takeaways

Animal cells need DMEM-type media with serum or defined supplements, 37 °C and 5% CO₂; preservation is slow freezing with 5–10% DMSO and storage in liquid nitrogen.
Anchorage-dependent cells grow as monolayers and are scaled on microcarriers (small beads give more area per volume); suspension-adapted cells like CHO and hybridomas scale far more easily.
Hybridomas fuse B cells with HGPRT-deficient myeloma and are selected in HAT (only the hybrid has both HGPRT and immortality); iPS cells are reprogrammed by Oct4, Sox2, Klf4 and c-Myc.
SCNT (Dolly) reprograms a somatic nucleus in an enucleated egg, keeping the egg’s mitochondria; knock-outs and knock-ins use homologous recombination at a defined locus, Cre–lox giving conditional control.
Microbes make primary (growth-associated) and secondary (idiophase) products — biofuels, PHB bioplastic, enzymes, antibiotics; recombinant protein host choice trades cost against glycosylation (E. coli < yeast < CHO).

Practice questions (15)

Attempt each one before opening the answer. Every explanation names the tempting wrong option as well as the right one, because that is where marks are lost.

In hybridoma technology, fused cells are grown in HAT medium. Aminopterin in HAT forces cells to use the nucleotide salvage pathway, and the surviving hybridomas owe their survival to
1. HGPRT contributed by the B-cell partner, with immortality from the myeloma
2. HGPRT contributed by the myeloma, with immortality from the B cell
3. resistance to aminopterin coded on a plasmid
4. their ability to grow without any nucleotides
Show answer
Answer: A — HGPRT contributed by the B-cell partner, with immortality from the myeloma
The myeloma is chosen HGPRT-deficient, so it cannot use the salvage pathway and dies in HAT when the de novo route is blocked; the B cell supplies functional HGPRT but cannot divide forever. Only the hybrid has both — the B cell’s HGPRT to survive HAT and the myeloma’s immortality to proliferate.
Microcarriers are used in bioreactors mainly to
1. provide a large attachment surface so anchorage-dependent cells can be grown in a stirred suspension
2. supply oxygen directly to the cells
3. remove carbon dioxide from the medium
4. act as a nitrogen source
Show answer
Answer: A — provide a large attachment surface so anchorage-dependent cells can be grown in a stirred suspension
Anchorage-dependent cells must attach to a surface, and a plain tank offers little area per volume; microcarrier beads give a large total surface that can be suspended and stirred, turning a surface-limited monolayer process into a scalable stirred-tank one. They are not an oxygen, CO₂ or nitrogen device.
A calf is produced by somatic cell nuclear transfer, taking a skin-cell nucleus from cow A and an enucleated egg from cow B, with cow C as surrogate. The calf’s mitochondrial DNA comes from
1. cow B, the egg donor
2. cow A, the nucleus donor
3. cow C, the surrogate
4. a mixture of A and C
Show answer
Answer: A — cow B, the egg donor
Mitochondria are cytoplasmic, so they come with the egg — from cow B, the egg donor. The nuclear genes come from cow A (the nucleus donor) and the surrogate C only gestates the embryo. This is why an SCNT clone is not a perfect genetic copy of the nucleus donor.
Which dye and counting device are used together to measure the viable cell count of an animal cell suspension?
1. trypan blue, which stains only dead cells, on a haemocytometer
2. Gram stain on an agar plate
3. ethidium bromide on an electrophoresis gel
4. phenol red in a spectrophotometer
Show answer
Answer: A — trypan blue, which stains only dead cells, on a haemocytometer
Live cells exclude trypan blue because their membranes are intact, so under a haemocytometer the blue cells are the dead ones and the viable fraction is the unstained count over the total. Gram stain and agar plates are for bacteria, ethidium bromide stains DNA on gels, and phenol red is the medium’s pH indicator.
A fermentation consumes 40 g per litre of glucose and produces 16 g per litre of ethanol. The ethanol yield on substrate Y(p/s) is ______ g per g (round off to two decimal places).
Numerical answer — type the value.
Show answer
Answer: 0.40
Y(p/s) = ethanol formed / glucose consumed = 16/40 = 0.40 g per g. This is 0.40/0.51 = 78% of the theoretical maximum ethanol yield of 0.51 g per g glucose. Dividing by the ethanol instead of the glucose, or using the cell yield, are the usual slips.
Why is CHO (Chinese hamster ovary) the standard host for producing therapeutic monoclonal antibodies rather than E. coli?
1. CHO performs human-like glycosylation and secretes correctly folded antibodies, which E. coli cannot
2. CHO grows faster and cheaper than E. coli
3. E. coli cannot replicate plasmids
4. CHO needs no bioreactor
Show answer
Answer: A — CHO performs human-like glycosylation and secretes correctly folded antibodies, which E. coli cannot
Antibodies are glycoproteins whose correct function and half-life depend on human-like glycosylation and proper folding and secretion — which mammalian CHO cells do and E. coli does not (E. coli lacks glycosylation and often makes inclusion bodies). E. coli is actually cheaper and faster; it is simply the wrong tool for a glycosylated secreted protein.
Microcarriers of 200 micrometre diameter are replaced by carriers of 100 micrometre diameter at the same total packed-bead volume in a bioreactor. By what factor does the total available attachment surface area increase? ______
Numerical answer — type the value.
Show answer
Answer: 2
For spheres, surface area per unit packed volume scales as 1/diameter (total area ∝ (number ∝ 1/d³) × (area per bead ∝ d²) = 1/d). Halving the diameter from 200 to 100 micrometre therefore doubles the area, a factor of 2. Treating area as 1/d² (giving 4) forgets that the number of beads also changes with volume.
A conditional knock-out using the Cre–lox system allows a gene essential for embryonic development to be studied in the adult because
1. Cre recombinase, expressed only in a chosen tissue or time, deletes the loxP-flanked gene only there and then
2. the gene is deleted in every cell from the zygote onward
3. loxP sites increase the gene’s expression
4. Cre protects the gene from mutation
Show answer
Answer: A — Cre recombinase, expressed only in a chosen tissue or time, deletes the loxP-flanked gene only there and then
In a conditional knock-out the gene is flanked by loxP sites but stays functional until Cre recombinase is present. By driving Cre from a tissue-specific or inducible promoter, the gene is deleted only in the chosen place or time, so an animal that would die as an embryo from a whole-body knock-out develops normally and the gene’s adult role can be probed.
Which of the following are correctly paired product and type? Select all that apply.
1. Penicillin — secondary metabolite (non-growth-associated)
2. Ethanol — primary metabolite (growth-associated)
3. Polyhydroxybutyrate — a microbial storage bioplastic
4. Citric acid — an antibiotic from Streptomyces
Show answer
Answer: A — Penicillin — secondary metabolite (non-growth-associated); B — Ethanol — primary metabolite (growth-associated); C — Polyhydroxybutyrate — a microbial storage bioplastic
Penicillin is a secondary metabolite, ethanol a growth-associated primary metabolite, and PHB a storage polyester used as a bioplastic. The pairing of citric acid with an antibiotic from Streptomyces is wrong twice: citric acid is a primary metabolite (an organic acid) and is made by the fungus Aspergillus niger, not by Streptomyces, and it is not an antibiotic.
When recombinant human insulin is expressed in E. coli, a common downstream challenge is that the protein
1. accumulates in insoluble inclusion bodies that must be solubilised and refolded
2. is hyper-glycosylated and must be deglycosylated
3. is secreted into milk and must be separated from casein
4. integrates into the host chromosome and cannot be recovered
Show answer
Answer: A — accumulates in insoluble inclusion bodies that must be solubilised and refolded
High-level expression of a foreign protein in E. coli often deposits it as misfolded aggregates (inclusion bodies), which are recovered, solubilised with a denaturant and refolded in vitro. E. coli does not glycosylate at all (so hyper-glycosylation is impossible) and does not secrete into milk; expression plasmids are episomal, not chromosomal.
Mouse monoclonal antibodies are "humanised" before clinical use mainly to
1. reduce the patient’s immune response against the foreign (mouse) protein
2. make them bind antigen more tightly
3. allow them to be grown in E. coli
4. convert them from IgG to IgM
Show answer
Answer: A — reduce the patient’s immune response against the foreign (mouse) protein
A mouse antibody is itself a foreign protein, so a patient mounts an anti-mouse response that clears it and can cause reactions; replacing mouse framework regions with human ones (chimaeric, humanised or fully human antibodies) reduces this. Humanisation is about immunogenicity, not affinity, host range or antibody class.
In screening soil for new antibiotic producers, an isolate is often judged a candidate on a primary screen by
1. a zone of growth inhibition it produces around itself on a lawn of a test organism
2. its ability to ferment lactose
3. its Gram-stain colour alone
4. the speed at which it grows
Show answer
Answer: A — a zone of growth inhibition it produces around itself on a lawn of a test organism
A primary screen for antibiotic activity looks for a clear zone where the isolate has inhibited a test organism around it (crowded-plate or agar-overlay). Lactose fermentation, Gram colour and growth rate say nothing on their own about whether the isolate secretes an inhibitory compound.
Fetal bovine serum is added to many animal cell media chiefly to supply
1. growth factors, hormones, attachment and carrier proteins
2. the main carbon source
3. the CO₂/bicarbonate buffer
4. antibiotics against contamination
Show answer
Answer: A — growth factors, hormones, attachment and carrier proteins
Serum provides the growth factors, hormones, attachment factors and carrier/binding proteins that cells need but basal medium lacks; glucose and glutamine are the carbon sources in the basal medium, bicarbonate is the buffer, and antibiotics are added separately. The drive toward serum-free media is to replace exactly these ill-defined serum components with defined ones.
Which of the following are features of making a transgenic animal by pronuclear microinjection? Select all that apply.
1. DNA is injected into a pronucleus of a fertilised egg
2. The transgene integrates at a random site, often in multiple copies
3. Integration is targeted to a chosen locus by homologous recombination
4. It can be used to produce a therapeutic protein in milk (gene pharming)
Show answer
Answer: A — DNA is injected into a pronucleus of a fertilised egg; B — The transgene integrates at a random site, often in multiple copies; D — It can be used to produce a therapeutic protein in milk (gene pharming)
Pronuclear microinjection puts DNA into a pronucleus, where it integrates randomly, usually as multiple copies, and is a standard way to make animals that secrete a protein into milk. Targeting to a defined locus by homologous recombination is gene targeting (knock-out/knock-in), a different method, so the claim that integration is targeted to a chosen locus does not describe microinjection.
Induced pluripotent stem (iPS) cells are produced by
1. reprogramming adult somatic cells with factors such as Oct4, Sox2, Klf4 and c-Myc
2. fusing B cells with myeloma cells
3. removing the nucleus from an egg
4. culturing the inner cell mass of a blastocyst
Show answer
Answer: A — reprogramming adult somatic cells with factors such as Oct4, Sox2, Klf4 and c-Myc
iPS cells (Takahashi and Yamanaka) are adult cells forced back to pluripotency by a defined set of reprogramming transcription factors, avoiding the use of embryos. B-cell/myeloma fusion makes hybridomas, enucleation is a step of SCNT, and culturing the inner cell mass gives embryonic (not induced) stem cells.