Recombinant DNA technologybiotka.mol.uj.edu.pl/...Medical_biotechnology_2015.pdf · Recombinant DNA...

Recombinant DNA technology Continued

Prof. Jozef Dulak Dr Urszula Florczyk

Web: www.biotka.mol.uj.edu.pl/zbm

Lecture 3 – 31 March 2015

Milestones of medical biotechnology

Edward Jenner's first vaccination

1797 – Edward Jenner inoculates a child with a viral vaccine to cowpox

(pus from cowpox blisters) to protect him from smallpox.

It took almost 200 years (till 1979) to eradicate smallpox from the world…

Vaccines

Antibiotics

1928 - Penicillin discovered as an antibiotic: Alexander Fleming.

1942 - Penicillin mass-produced in microbes.

1944 - Waksman isolates streptomycin, an effective antibiotic for tuberculosis.

a biological preparations that provide active acquired immunity to a particular disease

•  Vaccines stimulate the immune system to produce specific antibodies („soldiers”) to fight the specific virus or bacteria vaccinated for, if it ever attacks the body.

•  The ingredients of the vaccine are eliminated from the body within 24 to 48 hours so no long term side effects need be worried about.

•  Some vaccines contain live or attenuated viruses/bacteria and are not suitable for immune compromised individuals.

•  Vaccination programmes preventing polio, measles, mumps, rubella, varicella, rotavirus, hepatitis and many more have saved millions of lives and are advocated and supported by the World Health Organisation (WHO).

How do vaccines work?

http://www.everything-i-can.co.za/how-do-vaccines-work.html

Conventional vaccines

1.  Live attenuated organisms (non-pathogenic but immunogenic) – such as the Sabin oral polyomyleitis, measles and rubella vaccines

2. Inactivated (killed) microorganisms – eg. Salk parenteral poliomyeltis vaccine 3. Subunit vaccines – i.e. one or more antigenic components, as with influenza and

recombinant hepatitis B vaccines

4. DNA vaccines – delivery of nucleic acid encoding the pathogenic antigens

DNA vaccines

Vaccines

RJ Trent – Molecular Medicine, Academic Press 2012

Recombinant DNA technology for vaccines production

J. Pongracz, M. Keen – Medical biotechnology, Elsevier 2009

Advantage of higher production yield, incresed safety, constant and reproducible quality The encoding DNA sequence is inserted into an expression vector, most often Escherichia coli, yeast or a mammalian cell line The cells are cultured in vitro in bioreactors Product is purified from the supernatant or directly from the cells e.g. Vaccine against human papillomavirus (HPV)…

Modern subunit vaccines are produced as recombinant proteins

Human papillomavirus (HPV)

http://www.everything-i-can.co.za/how-many-are-infected.html

•  There are over 100 different types of HPV, and 40 of them affect the genital area •  For most women and girls the virus goes away on its own or it can develop into cervical cancer, precancerous lesions, or genital warts, depending on the HPV type. •  HPV is easy to transmit! HPV is spread mainly by direct skin-to-skin contact

Fact 74 % of new HPV infection occur in people aged 15 to 24, and 80 % of all women will have the virus at some point in their lives.

Human Papillomavirus (HPV) is a very common virus 8 out of 10 adults will come into contact with HPV.

Worldwide estimates on the burden of HPV & related genital diseases in women


-  There are over 100 different types of HPV, and 40 of them affect the genital area -  HPVs 16 and 18 are classified as high risk HPVs causing cervical cancer (about 70%cervical cancer cases) -  HPVs types 6 and 11 cause about 90% of genital wart cases

Human papillomavirus (HPV) and cervical cancer

-  The second most prevalent cancer among women -  Can have several causes – an infection with some type of human papillomavirus (HPV) is the greatest

risk. Unlike other cancers, cervical cancer is not passed down through family genes.

Cervical cancer


How the HPV vaccines are produced?

http://www.everything-i-can.co.za/how-do-vaccines-work.html

Produced by recombinant DNA technology, they are both effective and safe, posing no risk of infection or cancer Vaccines against HPV contain viruslike particles (VLPs)—empty viral capsids with no viral DNA inside.

Approved anti-cancer vaccines (1)

Cervical cancer The U.S. Food and Drug Administration (FDA) has approved two vaccines, Gardasil® and Cervarix®, that protect against HPV types 16 and 18, that cause ~70% of all cases of cervical cancer worldwide. In addition, Gardasil protects against infection by HPV types 6 and 11, which cause an estimated 90% of genital warts cases

Gardasil®, Merck (approved in June 2006); recombinant human papillomavirus vaccine [types 6, 11, 16, 18]

Cervarix™, GlaxoSmithKline (approved in Australia in May 2007, EU - September 2007)

In December 2014, the FDA approved a nine-valent Gardasil-based vaccine, Gardasil 9, to protect against infection with the strains covered by the first generation of Gardasil as well as five other HPV strains responsible for 20% of cervical cancers (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58).

http://www.cancer.gov/cancertopics/causes-prevention/vaccines-fact-sheet National Cancer Institute

Wikipedia

Approved anti-cancer vaccines (2)

Liver cancer

One of the major cause is the hepatitis B virus (HBV) infection - Two billion people worldwide are infected - 350 milion become chronic carriers - Important occupational hazard for health workers - Approximately 600 000 die annually from complications, such as cirrhosis and

hepatocellular carcionam - HBV is 50-100 times more infectious than HIV

Anti-hepatitis B virus vaccine – approved in 1981 – first anti-cancer vaccine - recombinant anti-HBV vaccine released in 1987

Today, most children in the USA are vaccinated against HBV shortly after birth

http://www.cancer.gov/cancertopics/causes-prevention/vaccines-fact-sheet National Cancer Institute RJ Trent – Molecular Medicine, Academic Press 2012

Gene replacement, gene knockout, and gene addition

Although genetic mapping and comparative sequencing projects can identify associations between DNA sequence and a disease or human trait , it remains necessary to test this link in relevant model to develop therapies -> investigative and translational biomedical research in mouse models

Recombinant DNA technology for understanding the mechanisms of diseases and development of innovative therapies

Having identified a target gene, a strategy must be developed by which the function of that gene can be repaired

Prerequisites for the creation of a mouse model

o  homologous genes must be identified between human and mouse (more than 98% genes are shared)

o  measurable biological parameters must be able to be compared between human and mouse (measurements of heart and lung function, blood pressure in freely moving animals, magnetic resonance imaging etc)

o  genetic changes in a specific gene in the mouse must result in a phenotype that correlates with the disease in humans

Genetic modifications in animals

Applied to test:

1. Single gene defects: both inherited and acquired 2. Multigene defects 3. Chromosomal defects

A mouse model have limitations when:

1. homologous genes do not exist between mouse and man 2. the gene defect does not have the same effect in mouse and man eg. Rb gene, HPRT gene 3. Physiological differences between mice and human: eg. apoE deficient mice and plaque development – different than in human

Transgenic animals

1.  Investigation of the mechanisms of disease 2.  Testing new therapies 3.  Producing new drug

Transgenic animals are genetically modified organisms (GMOs) which their genetic material changed

First transgenic mice

Palmiter et al., Nature 1982: 300: 611-615

Biochemistry. 5th edition. Berg JM, Tymoczko JL, Stryer L. New York: W H Freeman; 2002.

A DNA fragment containing the promoter of the mouse metallothionein-I gene fused to the structural gene of rat growth hormone was microinjected into the pronuclei of fertilized mouse eggs. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. implications for studying the biological effects of growth hormone, as a way to accelerate animal growth, as a model for gigantism, as a means of correcting genetic disease, and as a method of farming valuable gene products

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TransgenicAnimals.html

Transgene can be inserted into mouse genome either randomly or it may be incorporated in the chosen locus inside the mouse genome. The second strategy is called targeted approach and it is based on the homologous recombination.

Transgenic mice can be obtained by two major ways: -  transfer of cloned DNA into fertilized oocytes and cells from very early stage embryos -  transferring the transgene into cultured embryonic stem cells.

Two methods of producing transgenic mice

Strategies for creation of mouse models of human diseases

1. Disease-driven, directed genetics – a human mutation is identified and then a specific mouse model is made to mimic it

Type of genetic modifications: a. transgenesis – integration of DNA sequences randomly into the genome

b. gene targeting – precise modification by homologous recombination– gene is introduced into its normal genomic location: „knock-out”, „knock-in”

c. chromosome engineering

2. Mutagenesis-driven, non-directed genetics – relies on the

selection of disease phenotypes following random mutagenesis, induced by chemicals or gene trapping

eg. Mdx mice (Duchenne muscular dystrophy_ db/db mice – mice lacking leptin receptor

Transgenesis – integration of DNA sequences randomly into the genome

-  heritable integration

Method: pronuclear injection of fertilized mouse oocytes with the linearized DNA transgene

J Pongracz, M. Keen - Medical Biotechnology, 2009

- - random integration can lead to inadequate expression pattern

Large numbers of oocytes are produced from immature female mice by administering hormones which lead to superovulation The superovulated female is then mated and one-cell stage embryos are collected.

one-cell stage embryos are injected with the transgenic DNA into the large pronucleus

Conventional transgenes

•  A promoter •  An intron element to mimic normal gene structure •  The coding sequence of the molecule under investigation •  A polyadenylation signal

Jazwa et al. Gene 2013 J Pongracz, M. Keen - Medical Biotechnology, 2009

An alternative method for introducing foreign genes or targeted gene sequences into mice is to use

murine embryonic stem (ES) cells

What are embryonic stem cells?

pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo - develop from eggs fertilized in vitro -  derived from 4-5 days old embryos - isolated from ~ 8 cell embryo of inner cell mass

Kaur et al., Journal of Diabetology, 2012

Murine embryonic stem cells

Sir Martin Evans, Mario Capecchi, Olivier Smithies

Nobel Prize 2007 for their discoveries of principles for introducing specific gene modifications in mice by the

use of embryonic stem cells

Wikipedia

The transgenic mouse generation by the method with the use of embryonic stem cells involves:

-  the ES cell cultivation in vitro -  DNA introduction usually by electroporation -  positive clone selection due to the presence of the selectable marker gene

in the introduced DNA

Difficulties can be connected with the random transgene integration event and with the germline transmission of the transgenic ES cells

The method is commonly used for the DNA integration by homologous recombination in order to obtain knockouts and knockins

– GENE TARGETING

The transgenic mouse generation by the method with the use of embryonic stem cells

Gene targeting - precise modification

-  enables the removal or replacement of specific gene by homologous recombination -  key – EMBRYONIC STEM CELLS (ES cells) Method:


- once reimplanted into the uterus of a pseudo-pregnant foster mother, the injected „donor” ES cells compete with cells in the host blastocyst to form the developing embryo and ultimately lead to a chimeric mouse

-  if the germ cells of the chimera also contain cells derived from the donor ES cells, some progeny resulting from mating will have one set of chromosomes derived completely from the donor , thereby establishing a „line”

genetically modified ES cells


Gene targeting – gene knock-out

Most widely applied use of homologous recombination in ES cells has been the generation of knockout mice - Usually all or part of the protein coding sequences is deleted or an insertion is made into a exon encoding an essential domain

- - the targeting vector is introduced into ES cells by subjecting cell suspension in a solution of DNA to a short electric pulse

- - ES cells that succesfully exchange the targeting sequences by homologous recombination for one of its two corresponding genomic sequences are able to grow in the presence of the selective antibiotic - ES clone is then injected into blastocysts to ptoduce a chimera

Gene knockout technology

Molecular biology of the gene

Knockout of VEGF is lethal in heterogenous form

Ferrara & Allitalo, Nature medicine, 2000

Gene targeting – conditional gene deletion Use of cre recombinase for conditional knockouts

Most widely used: Cre recombinase and its 32 base recognition element, loxP A gene is engineered by homologous recombination in ES cells so that the whole gene or an exon encoding crucial protein domain , is flanked by recognition sites for a recombinase enzyme that can delete the intervening sequences

Molecular biology of the gene

Gene knockout is restricted by expressing the recombinase

in specific tissueas or at particular time

Jazwa et al. Gene 2013

Gene targeting - knock-in

- The majority of diseases with genetic basis involve small sequence changes - The same ES-based approaches can be used to create mouse lines to model such mutations – so called „knock-in”

Small mutational changes (asterisk) can be introduced by targeting and usually it is desirable to remove the selection cassette by recombinase system


Human apoE is a 299 amino acid protein that occurs in three major isoforms (apoE2, apoE3 and apoE4) encoded by three APOE alleles (ε2, ε3 and ε4) differing with respect to the presence of cysteine or arginine at two polymorphic sites.

•  ApoE3, the most

common isoform, has cysteine at amino acid position 112 and arginine at 158

•  ApoE2 has cysteine at both 112 and 158

•  ApoE4 has arginine at both sites

LDLR binding

Lipids binding

A multifunctional glycosylated protein, mostly involved in the transports of lipoproteins and cholesterol

Apolipoprotein E

-  The most common allele is e3, which is found in more than half of the general population.

-  ApoE4 preferentially binds to lower density lipoproteins and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer Disease

-  The APOE e2 allele has been shown to greatly increase the risk of a rare condition called hyperlipoproteinemia type III.

Polymorphism of apolipoprotein E

ApoE exerts its biological functions, especially lipid transportation, by binding to its receptors, the low-density lipoprotein receptor (LDLR) family.

There is only one isoform of apoE in rodents. Murine apoE preferably associates with HDL, and its

clearance is mainly through LDLR

In human:

En face preparations of oil red O stained aortas from C57BL6/J and ApoE KO mice at 26 weeks of age (from Behr-Roussel et. Al, 2006).

The Apolipoprotein E knockout mouse model is one of the most widely used experimental model of atherosclerosis. These mice rapidly develop atherosclerotic lesions that resemble human lesions evolving over time from initial fatty streaks to complex lesions. PATHOPHYSIOLOGICAL FEATURES Cardiovascular features: • Apolipoprotein E deficiency directly results in the increase of plasma levels of LDL and VLDL. •  Very high cholesterol level (500-600 mg/dl instead of 50-100 mg/dl) • Spontaneous development of atherosclerotic lesions throughout the arterial tree appearing first in the aortic arch in young mice and progressing in the thoracic and abdominal aorta in older mice

Apolipoprotein E knockout mouse

Maeda et al., Atherosclerosis 2007

•  Homozygous for a human APOE4 (or APOE3 or APOE2) gene targeted replacement of the endogenous mouse Apoe gene •  Expresses human apoliprotein E4 isoform under the control of the murine Apoe regulatory sequences

To assess allele-specific differences in apoE function in vivo, it is desirable to have mouse strains in which the murine apoE is replaced by human apoE

isoforms expressed under the natural regulation of this protein.

KNOCK-IN technology

RIKEN BioResource Center (BRC)

Polymorphism of apoE – knock-in technology

Targeting strategy for apoE4 knock-in mice and homologous intergration of the transgene. (A) Schematic diagram of the knock-in targeting strategy. (Top) The structure of the endogenous Apoe locus including exons 1–4 (black boxes). (Middle) The targeting vector containing the human apoE4 cDNA (hu cDNA). (Bottom) The predicted structure of the knock-in allele after homologous recombination. The neomycin-resistance (neo) and thymidine kinase (TK) genes are for selection of the targeted ES cells. The neo cassette is flanked by 34 bp loxP sequences (triangles). pA represents the endogenous polyadenylation signals. Restriction sites: B, BglII; E, EcoRI; H, HindIII; N, NcoI; S, SalI; X, XmnI. (B) Southern blot analysis of tail-tip DNA from wild-type (+/+), heterozygous (4/+) and homozygous (4/4) knock-in mice digested with HindIII and hybridized with the 3′ probe shown in (A). The wild-type Apoe allele generates an 8.0 kb HindIII fragment, whereas the targeted allele yields the diagnostic 6.4 kb HindIII fragment.

Hamanaka et al., Hum Mol Genet. 2000; 9(3): 353-361

Generation of human apoE4 knock-in mice – similar strategy for ApoE3 and ApoE2


1. Disease-driven, directed genetics – a human mutation is identified and then a specific mouse model is made to mimic it Type of genetic modifications:

a. transgenesis – integration of DNA sequences randomly into the genome b. gene targeting – precise modification – gene is introduced into its normal genomic location: „knock-out”, „knock-in” c. chromosome engineering




•  Aberrations in human chromosome copy number and structure are common and deletorious, both as development-associated defects and as aqcuired events in tumors (e.g. trisomy of chromosome 21 in Down syndrome)

•  The combination of gene targeting in ES cells, recombinase technology and other techniques makes it possible to generate new chromosomes carrying specific and defined deletions, duplications, inversions and translocations, which can serve as models for human chromosomal aberrations

Chromosome engineering

Cre-loxP technology can be used: - To create very large deletions or inversions on a single chromosome -  To bring about specific recombination between two different

chromosomes

The Metabolic and Molecular Basis of Inherited Diseases, McGraw Hill, 2001

Summary- Strategies for generating knockout/transgenic mice


1. Disease-driven, directed genetics – a human mutation is identified and then a specific mouse model is made to mimic it Type of genetic modifications:

a. transgenesis – integration of DNA sequences randomly into the genome b. gene targeting – precise modification – gene is introduced into its normal genomic location c. chromosome engineering




Mutagenesis-driven, non-directed genetics

This approach requires selection of phenotypes following either spontaneous or induced random mutation

Random mutagenesis strategies can be divided based on mutagen used:

- Gene trapping using transgene insertion as the mutagen

- Chemical mutagenesis

Alkylating mutagen (N-ethyl-N-nitrosourea) -  ENU-treated males are mated with females, progeny are put into a breeding programme -  Phenotypic screening

Gene trapping using transgene insertion

Reporter gene is activated following insertion into an endogenous transcription unit -  Gene trap vectors contain a splice acceptor sequence upstream of a reporter and are activated following insertions into introns - - prevents expression of gene sequence downstream of the site of insertion

Based on the pattern of reporter expression in vitro, ES cell clones are tested in vivo: o  For expression of the reporter in chimeric embryos following introduction into blastocysts o  then, for both reporter expression and function of the trapped gene in embryos and adults, if germline transmission can be achieved

Drug testing using mouse models

e.g. If a drug candidate is thought to operate through a particular gene product this can be tested by comparing the effect of the compound in wild type and mice mutated at the relevant gene locus

DRUG DISCOVERY

Also to study in vivo efficacy of candidate compounds – mice can be humanized with respect to small subgroups of proteins responsible for the major routes of transport and metabolism of a large proportion of drugs

Dulak et al. Circulation 2008

Heme oxygenase-1

HO-1 Fe2+

antioxidant anti-inflammatory anti-apoptotic pro-angiogenic cytoprotection

ACTIVITY PRODUCT MECHANISM

ferritin synthesis iron ATP-ase pump

BVR

activation of sGC leading to cGMP production

p38 MAPK regulation

BILIVERDIN

BILIRUBIN

CO

ROS scavenging Inhibition of complement

anti-apoptotic anti-proliferative anti-thrombotic anti-inflammatory pro-angiogenic cytoprotection

antioxidant anti-inflammatory anti-apoptotic cytoprotection

HEME

others

Investigation on the role of heme oxygenase-1 in human diseases

1.  Inducers of heme oxygenase-1 expression/activity 2. Inhibitors of Hmox-1 activity

A) small molecular compounds b) siRNA

3. Heme oxygenase-1 knockout mice 4. Heme oxygenase-1 transgenic mice

SnPP s.c. SnPP i.p. PBS

Day 0

Day 3

Day 7

Wound healing is dependent on HO-1

Lack of HO-1 impairs blood vessel formation in wounds

Deshane et al., J Exp Med., 2007, Grochot-Przeczek et al. , PLoS ONE 4(6): e5803; 2009

Inhibition of HO-1 attenuates wound healing Lack of HO-1 attenuates wound healing

Development of stable transgenic mouse line overexpressing HO-1 in the skin

To study skin related processes,such as wound healing, cancerogenesis, psoriasis…

Modified from: Human Molecular Genetics 2 2nd ed. New York and London: Garland Science; c1999.

ES cell- based, random DNA insertion strategy

ES cell cultivation in vitro, DNA introduction by electroporation and positive clone selection due to the presence of the selectable marker gene in the introduced DNA. After the amplification and confirmation of the transgene presence, ES clones are microinjected into mouse blastocyst. The founders derived from the embryos that have undergone the transgene integration will be chimeric, composed of a mixture of transgenic ES cell and wild type host cells. If the transgenic ES cells have contributed to the germ line of chimeric mice, their offspring can be fully heterozygous and after crossing these heterozygotes to each other, can develop homozygous mice strain.

From ES cells to transgenic mice: microinjection of ESC into the murine blastocyst

3 lines of transgenic mice:

•  K14HO-1 clone 9

•  K14 HO-1 clone 78

•  K14 HO-1 clone 94 Germline

transmission

Random insertion into the genome results in an indiscriminate transgene incorporation into chromosomes and that strategy is commonly used for generation of transgenic mice which overexpress the gene of interest.

Genotyping by PCR and Southern blotting

PCR with primers specific for human HO-1

Southern blotting with probe specific for human HO-1

Higher expression of HO-1 in epidermis of KER14-HO1 mice

WT Tg

HO-1+/+ HO-1Tg Skin

Higher expression of HO-1 in primary murine keratinocytes of KER14-HO1 mice

Primary murine keratinocytes culture: 2 days after isolation

Tg Wt Tg Wt Tg

Grochot-Przeczek et al. , PLoS ONE 4(6): e5803; 2009

HO-1 is required for blood vessel formation

Grochot-Przeczek et al. , PLoS ONE 4(6): e5803; 2009

Skin wound healing

0 10 20 30 40 50 60 70 80 90

100

0 1 2 3 4 5 6 7 8 10 11 12 13

Wou

nd

clo

sure

[% o

f d

ay 0

]

HO-1 Tg HO-1 WT

HO-1-HT HO-1-KO

Day after wounding

Inhibition of HO-1 attenuates wound healing, while its overexpression in the skin promotes it

Transgenic and knockout mice are indispensable tools for investigation the mechanisms of diseases and effectiveness

of therapies

Transgenic animals for drug production

Orphan diseases and recombinant DNA technology

Orphan disease - a disease that has not been „adopted” by the pharmaceutical industry because it provides little financial incentive for the private sector to make and market new medications to treat or prevent it. An orphan disease may be a rare disease (according to US criteria, a disease that affects fewer than 200,000 people) or a common disease that has been ignored (such as tuberculosis, cholera, typhoid, and malaria) because it is far more prevalent in developing countries than in the developed world.

• ATryn – first drug produced by transgenic goats, which has been registered by the European Commission in July 2006

•  The FDA approval (2009) of the first biological product produced by genetically engineered animals recombinant human antithrombin.

Because hereditary AT deficiency occurs in a small population (approximately 1 in 5,000 people in the United States), the FDA granted ATryn an orphan drug designation.

One GM goat can produce the same amount of antithrombin in a year as 90,000 blood donations

Transgenic animals as drug factories

An anticoagulant, human anti-thrombin - a natural serum protein with anti-thrombotic and anti-inflammatory properties.

used for the prevention of blood clots in patients with a rare disease known as hereditary antithrombin (AT) deficiency;

It is used in obstetrics, treatment of deep vein thrombosis from the milk of goats

http://pharmacologycorner.com/fda-approves-atryn-recombinant-human-antithrombin-for-the-treatment-of-hereditary-antithrombin-deficiency/

Atryn – first approval for a biological product produced by genetically engineered animal

Biotechnology - achievements

Biotechnology has created:

* more than 200 new therapies and vaccines, including products to treat cancer, diabetes, AIDS and autoimmune disorders.

* more than 400 drug products and vaccines currently in clinical trials targeting more than 200 diseases, including various cancers, Alzheimer’s disease, heart disease, diabetes, multiple sclerosis, AIDS and arthritis.

* hundreds of medical diagnostic tests for early detection of diseases, for keeping the blood supply safe, or for detection of pregnancy at home.

* DNA fingerprinting, which has dramatically improved criminal investigation and forensic medicine.

Biotechnology therapeutics approved by the U.S. Food and Drug Administration (FDA) to date are used to treat many diseases, including leukemia and other cancers, anemia, cystic fibrosis, growth deficiency, rheumatoid arthritis, hemophilia, hepatitis, genital warts, and transplant rejection.

Next lecture

Gene therapy

14 April, 2015

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