Cell Potency
Cells are classified by their potential to differentiate into different cell types. This potential,
known as potency, varies:
• Totipotent cells are the most versatile, capable of producing all cell types of the
developing organism, which includes both the embryonic tissues and
extraembryonic tissues like the placenta.
• Pluripotent cells are slightly more restricted. They can make all cells of the
embryo proper, including germ cells and cells from any of the three germ layers
(ectoderm, mesoderm, endoderm). Thus, they can make any cell of the body, but
not the extraembryonic tissues.
• Multipotent cells have a more limited differentiation potential, as they can only
make cells within a given germ layer. For instance, multipotent stem cells from
blood (a mesodermal tissue) can produce all blood cell types but cannot produce
neural cells (ectoderm) or liver cells (endoderm).
• Unipotent cells are the most specialized, capable of making cells of only a single
cell type. An example is a germ cell stem cell which gives rise to cells that mature
into egg or sperm, but no other cell types.
Production of Recombinant Pharmaceuticals
Genetic engineering has been extensively applied to produce pharmaceuticals. The
sources detail the production of several key recombinant proteins:
• Human Insulin: This was one of the earliest uses of recombinant technology,
performed by Genentech in 1978. Prior to this, bovine and porcine insulin were
used, but they often caused immunogenic reactions and presented purification and
contamination issues. To overcome these problems, researchers inserted human
insulin genes into a suitable vector, such as E.coli.
o The process involved synthesizing genes for the two insulin chains, A and B.
These genes were inserted into plasmids along with a strong promoter,
specifically a lacZ promoter. The genes were positioned such that the
insulin A and B chains would be linked to B-galactosidase residues,
separated by a methionine residue. This methionine residue allows for easy
separation of the insulin chains by adding cyanogen bromide.
o The engineered vector was then introduced into E.coli cells. Inside the
bacteria, the genes were "switched-on" by the bacteria to translate the
, genetic code into the protein chains. The purified insulin A and B chains
were then attached to each other through disulphide bond formation in the
laboratory. The process is illustrated to show the human insulin-producing
gene from a human pancreas cell being inserted into a bacterial plasmid,
creating recombinant DNA. This is then introduced into a bacterium, which
multiplies in a fermentation tank, producing human insulin for extraction and
purification.
• Human Growth Hormones: The proteins Somatostatin and Somatotrophin regulate
growth processes. Malfunction of these proteins can lead to disorders like
Acromegaly (uncontrolled bone growth) and Dwarfism. Somatostatin was the first
human protein synthesized in E. coli. Its short length of only 14 amino acids made
it well-suited for artificial gene synthesis. The strategy used was the same as for
recombinant insulin, involving insertion into a lacZ' vector, synthesis of a fusion
protein, and cleavage with cyanogen bromide.
• Interferons (IFNs): These are signaling proteins released by host cells in response
to pathogens or tumor cells. They cause nearby cells to heighten their anti-viral
defenses. Recombinant DNA technology has proven to be the most satisfactory
method for large-scale production of human interferons. Genes for all three types
of human IFNs (HuIFN) have been cloned in microorganisms and expressed.
Although HuIFN-beta and gamma produced this way lack the glycosylation found
in natural interferons, their specific activity is not affected.
• Recombinant Secondary Metabolites: The sources mention the importance of
antibiotics and how genetic engineering is used to increase their production. GM
micro-organisms are utilized for this purpose. Another technique to increase yields
is gene amplification, where copies of genes coding for enzymes involved in
antibiotic production are inserted back into a cell via vectors like plasmids.
• Monoclonal Antibodies: The sources also describe the production of monoclonal
antibodies using recombinant technology. This involves isolating antibody genes
(from hybridoma cells, B cells, or antibody libraries), cloning them into an
expression vector containing elements like a promoter, selectable marker, signal
peptide, and optional tags. This vector is then introduced into host cells such as
bacteria (E. coli), yeast, or mammalian cells (e.g., CHO cells). The chosen host
depends on the antibody format (bacteria for fragments, mammalian cells for full-
length IgG). The host cells are cultured to maximize antibody yield, followed by
purification using methods like affinity chromatography (Protein A/G) or other
Cells are classified by their potential to differentiate into different cell types. This potential,
known as potency, varies:
• Totipotent cells are the most versatile, capable of producing all cell types of the
developing organism, which includes both the embryonic tissues and
extraembryonic tissues like the placenta.
• Pluripotent cells are slightly more restricted. They can make all cells of the
embryo proper, including germ cells and cells from any of the three germ layers
(ectoderm, mesoderm, endoderm). Thus, they can make any cell of the body, but
not the extraembryonic tissues.
• Multipotent cells have a more limited differentiation potential, as they can only
make cells within a given germ layer. For instance, multipotent stem cells from
blood (a mesodermal tissue) can produce all blood cell types but cannot produce
neural cells (ectoderm) or liver cells (endoderm).
• Unipotent cells are the most specialized, capable of making cells of only a single
cell type. An example is a germ cell stem cell which gives rise to cells that mature
into egg or sperm, but no other cell types.
Production of Recombinant Pharmaceuticals
Genetic engineering has been extensively applied to produce pharmaceuticals. The
sources detail the production of several key recombinant proteins:
• Human Insulin: This was one of the earliest uses of recombinant technology,
performed by Genentech in 1978. Prior to this, bovine and porcine insulin were
used, but they often caused immunogenic reactions and presented purification and
contamination issues. To overcome these problems, researchers inserted human
insulin genes into a suitable vector, such as E.coli.
o The process involved synthesizing genes for the two insulin chains, A and B.
These genes were inserted into plasmids along with a strong promoter,
specifically a lacZ promoter. The genes were positioned such that the
insulin A and B chains would be linked to B-galactosidase residues,
separated by a methionine residue. This methionine residue allows for easy
separation of the insulin chains by adding cyanogen bromide.
o The engineered vector was then introduced into E.coli cells. Inside the
bacteria, the genes were "switched-on" by the bacteria to translate the
, genetic code into the protein chains. The purified insulin A and B chains
were then attached to each other through disulphide bond formation in the
laboratory. The process is illustrated to show the human insulin-producing
gene from a human pancreas cell being inserted into a bacterial plasmid,
creating recombinant DNA. This is then introduced into a bacterium, which
multiplies in a fermentation tank, producing human insulin for extraction and
purification.
• Human Growth Hormones: The proteins Somatostatin and Somatotrophin regulate
growth processes. Malfunction of these proteins can lead to disorders like
Acromegaly (uncontrolled bone growth) and Dwarfism. Somatostatin was the first
human protein synthesized in E. coli. Its short length of only 14 amino acids made
it well-suited for artificial gene synthesis. The strategy used was the same as for
recombinant insulin, involving insertion into a lacZ' vector, synthesis of a fusion
protein, and cleavage with cyanogen bromide.
• Interferons (IFNs): These are signaling proteins released by host cells in response
to pathogens or tumor cells. They cause nearby cells to heighten their anti-viral
defenses. Recombinant DNA technology has proven to be the most satisfactory
method for large-scale production of human interferons. Genes for all three types
of human IFNs (HuIFN) have been cloned in microorganisms and expressed.
Although HuIFN-beta and gamma produced this way lack the glycosylation found
in natural interferons, their specific activity is not affected.
• Recombinant Secondary Metabolites: The sources mention the importance of
antibiotics and how genetic engineering is used to increase their production. GM
micro-organisms are utilized for this purpose. Another technique to increase yields
is gene amplification, where copies of genes coding for enzymes involved in
antibiotic production are inserted back into a cell via vectors like plasmids.
• Monoclonal Antibodies: The sources also describe the production of monoclonal
antibodies using recombinant technology. This involves isolating antibody genes
(from hybridoma cells, B cells, or antibody libraries), cloning them into an
expression vector containing elements like a promoter, selectable marker, signal
peptide, and optional tags. This vector is then introduced into host cells such as
bacteria (E. coli), yeast, or mammalian cells (e.g., CHO cells). The chosen host
depends on the antibody format (bacteria for fragments, mammalian cells for full-
length IgG). The host cells are cultured to maximize antibody yield, followed by
purification using methods like affinity chromatography (Protein A/G) or other
