Biopharming Slide

Biopharming
 Biopharming is the production and use of transgenic

plants and animals genetically engineered to produce
pharmaceutical substances for use in humans or animals
 Also known as molecular farming or molecular

pharming.
 Most important utilization of transgenic animals

involving the target production of therapeutically
recognized proteins.
Transgenic animals as bioreactor
 They are Genetically modified by stable incorporation
of foreign DNA into their genome.
 Expressing recombinant proteins in milk, urine,

blood, sperm, or eggs, or even to grow organs for
transplantation.
 Having post translational modification machinery

(glycosylation, phosphorylation etc) essential for proper
folding and functioning of complex eukaryotic proteins.
Commonly used transgenic animals
 Rabbit
 Goat
 Sheep
 Pigs
 Cows
 Mice
Products
 Growth factors
 Hormones
 Antithrombin III
 α1- Antitrypsin
 Calcitonin
 Erythropoietin
 Fibrinogen
 Hemoglobin
 Human serum
albumin
Cont.
 Insulin
 α-Lactalbumin
 Human tissue plasminogen activator
 Lactoferrin
 Lysozyme
 Interferons
 Immunoglobulins
 Spider silk proteins
 Blood clotting proteins
Insulin-like growth factor 1 (IGF-1)
 Stable production of IGF-1in the milk of transgenic mice.
 The transgene gene consisting of the cDNA for human

IGF-1 and a mammary gland specific expression cassette
derived from bovine alpha-S1-casein sequences.
 Transgene expression was shown to be strictly tissue and

lactation period specific with the average production of
543 ± 41 µg/ml protein.
Human nerve growth factor (hNGF)
 hNGF is a clinically effective protein in the treatment
of many human neuronal and non-neuronal diseases.
 A transgenic mouse line has been generated carrying a

salivary gland specific promoter-driven hNGF transgene.
 This mouse line secretes high level of hNGF protein

in their saliva (1.36 μg/mL).
 The secreted hNGF induces neuronal differentiation

into PC12 cell line.
Human growth hormone
 Transgenic rabbit has been used to produce the hGH in

their milk.
 The expression pattern of recombinant human growth

hormone (rhGH) in transgenic rabbits consists of hGH
genomic sequences and the rat whey acidic protein (WAP)
promoter.
 Biologically active hGH was detected in the female

rabbit up to the level of 10 μg/ml.
Recombinant human antithrombin III
 Transgenic cloned goats are considered as the best

candidate for the production of recombinant human
antithrombin III (rhATIII).
 The transgene construct consists of the human ATIII

cDNA and enterokinase recognised peptide-DNA
sequence which are ligated to a goat beta-casein
promoter and poly (A) singling sequences, neomycin
selection gene is linked at the end of the poly (A)
singling region.
Cont.
 The transgenic cloned goat are derived from by the
nuclear transplantation of the goat fetal fibroblast which
is transfected by the transgene construct.
Human α1‐antitrypsin (α1AT)
 Transgenic sheep have a great potential for the

large scale production of α1AT.
 Gene construct comprises cDNA sequences

encoding human alpha‐1‐antitrypsin (α1AT) which is
inserted into the first exon of the ovine β‐lactoglobulin
gene (BLG).
 The recombinant α1AT protein are biologically

active and shows the same electrophoretic mobility as
human plasma‐derived α1AT
Recombinant human erythropoietin
 Erythropoietin (EPO) is a hormone produced primarily
by the kidneys plays a key role in the production of red
blood cells.
 A line of transgenic swine has been developed

harboring recombinant human erythropoietin (hEPO)
through microinjection into fertilized pig zygotes.
 The milk from the transgenic pigs has been analyzed

which produces a significant amount of hEPO protein
(877.9 ± 92.8 IU/1 ml).
Recombinant human fibrinogen
 Transgenic mice have been used for the high level
expression of r-human fibrinogen to the mammary gland.
 The transgene construct consists of three expression

cassettes, each containing the genomic sequence for one
of the three human fibrinogen chains which are controlled
by sheep whey protein beta-lactoglobulin promoter
sequences.
Cont.
 Polyacrylamide gel electrophoresis of milk of the
transgenic mice demonstrated the presence of human
fibrinogen subunits at concentrations of 2000
micrograms/ml
 Incubation of the transgenic milk with thrombin and

factor XIII resulted in a cross-linked fibrin clot,
indicating that a major portion of the secreted fibrinogen
was functional
Recombinant human serum albumin
 Transgenic cattle expressing high levels of recombinant

human serum albumin (HSA) in their milk may as an
alternative source for commercial production
 Cattle were genetically modified to express HSA in

milk using phiC31 integrase system and somatic cell
nuclear transfer (SCNT)
Cont.
 The mammary-specific expression plasmid pIACH(-),
containing the attB recognition site for phiC31 integrase,
were co-transfected with integrase expression plasmid
pCMVInt into bovine fetal fibroblast cells (BFFs).
 The phiC31 integrase system is an efficient and safety

gene delivery tool for producing HSA transgenic cattle
human plasminogen activator (rhPA)
 Expression efficacy of recombinant human
plasminogen activator in homozygous transgenic rabbits
is significantly very high.
 Homozygous transgenic rabbits were obtained using

an effective rhPA mammary‑specific expression vector
PCL25/rhPA
 The expression of rhPA in homozygous transgenic

rabbit was ~950 µg/ml, which was markedly higher in
comparison with that in hemizygote rabbits
human Interferon-γ
 Human interferon-γ (hIFN-γ) is a key cytokine
endowed with multiple biological activities such as
antiviral, antibacterial, antiparasitic, antiproliferative, and
immunomodulatory activity
 A transgenic mouse line has been generated for

expressing the hIFN-γ gene under the control of the
murine whey acidic protein (mWAP) gene promoter into
the mammary glands of lactating mice
spider silk
 Researchers from the University of Wyoming have
developed a way to incorporate spiders' silk-spinning
genes into goats, allowing the researchers to harvest the
silk protein from the goats’ milk for a variety of
applications.
 For instance, due to its strength and elasticity, spider
silk fiber could have several medical uses, such as for
making artificial ligaments and tendons, for eye sutures,
and for jaw repair. The silk could also have applications
in bulletproof vests and improved car airbags

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Biopharming

 Biopharming is the production and use of transgenic

 Also known as molecular farming or molecular

 Most important utilization of transgenic animals

 Expressing recombinant proteins in milk, urine,

 Having post translational modification machinery

 Stable production of IGF-1in the milk of transgenic mice.

 The transgene gene consisting of the cDNA for human

 Transgene expression was shown to be strictly tissue and

 A transgenic mouse line has been generated carrying a

 This mouse line secretes high level of hNGF protein

 The secreted hNGF induces neuronal differentiation

 Transgenic rabbit has been used to produce the hGH in

 The expression pattern of recombinant human growth

 Biologically active hGH was detected in the female

 Transgenic cloned goats are considered as the best

 The transgene construct consists of the human ATIII

 Transgenic sheep have a great potential for the

 Gene construct comprises cDNA sequences

 The recombinant α1AT protein are biologically

 A line of transgenic swine has been developed

 The milk from the transgenic pigs has been analyzed

 The transgene construct consists of three expression

 Incubation of the transgenic milk with thrombin and

 Transgenic cattle expressing high levels of recombinant

 Cattle were genetically modified to express HSA in

 The phiC31 integrase system is an efficient and safety

 Homozygous transgenic rabbits were obtained using

 The expression of rhPA in homozygous transgenic

 A transgenic mouse line has been generated for

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