Download Biotechnology: Principles, Applications, and Social Implications

Biotechnology
Gene therapy, Cloning
Heredity No 439
by RNDr. Hana Zoubková, PhD
What is Biotechnology?
The application of technology to improve a biological
organism by changing or adding genes from another
organism
…………...produce Genetically modified organisms
Gene engineering, transgenosis, transgenic organism
What is Cloning?
The process of producing populations of genetically
identical individuals
Molecular cloning – amplification of DNA fragments
Cell cloning – an application of stem cells
Organism cloning – somatic cell nuclear-transfer
What is Gene therapy?
The application of technology to improve (to heal)
a human organism by changing or adding genes
from another organism
Cloning of cell or organism
Reproductive vs. therapeutic cloning
An aim of reproductive cloning is origin of a baby
An aim of therapeutic cloning is to provide stem cells
for a patient, which requires a transplant
Technique of embryo division – old technique of formation
genetically identical individuals, division of morula or blastocyst
Technique of nucleus transfer a transfer of
somatic cell nucleus to enucleated egg
Stem cells
Embryonic stem cells – an application is forbidden
Infant and adult stem cells – an application is permitted
and they are used for therapy. They are present in small
numbers in
Bone marrow
Peripheral blood
Skin epithelium
Umbilical cord blood
Dental pulp of infant’s teeth
Stem cells may be obtained by reprogramming
somatic cells
Sources of adult and infant stem cells
Gene therapy
Somatic
A genome is changed, but the change is not passed
to other generation. The gene in patients cells are
repaired and returned back.
Germ gene therapy
A genome is changed directly in ovum or sperm cell
and the change is passed to other generations
- is not proceeded for any kind of animals
Researchers may use several approaches
A transgen may be inserted into a non-specific location within
the genome to replace a function of an abnormal gene.
An abnormal gene might be swapped for a normal gene through
homologous recombination.
An abnormal gene could be repaired through selective reverse
mutation, which returns the gene to its normal function.
An gene might be turned on or off, a regulation of its
expression is altered.
1
2
4
3
Ex vivo – pathologic tissue is taken from patient. Cells
accept transgen in laboratory conditions and
after that are cells given back to patient
In vivo – we prepare viral vectors with transgene and
we introduce (infect) patient‘s tissue with the
viral vectors
Problems:
Multifactorial disorders
Short-lived nature of gene therapy
Immune response
The genes are not expressed in intended tissues and in
intended place of the genome.
Examples of gene therapy trials for inherited disorders
Disorder
Cells altered
Gene therapy strategy
SCID - ADA deficiency
T cells and hemopoietic
stem cells
Ex vivo GAT using
recombinant retroviruses
containing an ADA gene
Cystic fibrosis
Respiratory epithelium
Familial
hypercholesterolemia
Liver cells
Gaucher's disease
glucocerebrosidase
Hemopoietic stem cells
In vivo GAT using
recombinant
adenoviruses or
liposomes to deliver the
CFTR gene
Ex vivo GAT using
recombinant retroviruses
to deliver the LDL
receptor gene (LDLR)
Ex vivo GAT using
retroviruses to deliver the
gene (GBA)
Molecular cloning - steps
Gene manipulation and introduction and multiplication
1. An identification and isolation of gene
2. We modify an existing gene (a new allele) or we create
gene knock-out or we modify foreign DNA or synthetic
genes to be transgene
3. The introduction of transgene into an organism for
multiplication
4. Selection of transgenic organisms and a usage of
multiplicated transgene or its protein
Molecular cloning: the principle of bacterial production of
insulin, it is used a vector with transgene (insulin gene)
1. Identification, isolating unknown gene by
Homology cloning based on a similarity to known
genes. e.g. Mouse‘s gene will help to determine the
localization of human one in hybridization method
Complementary genetics we predict nucleotide
sequences due to known aminoacids sequences
Map-based cloning based on searching of genetic
markers in linkage with the unknown gene – chromosome
walking
2. Preparation of recombinant DNA
which contains polylinker: artificially synthetized part of
DNA with specific spots for restriction endonucleases
(RE), which allow to incorporate gene of interest transgen,
also contains resistance to two or more antibiotics and
origin of replication. The whole recombinant DNA must
be short enough for vector.
● Gene of interest
● Selectable marker
● Insertion sequences
Isolated gene (gene of interest) becomes transgene - the
gene must be combined with other genetic elements in order
to be expressed properly. The gene can also be modified at
this stage for better expression or effectiveness.
Promoter TP
Coding Region
Promoter Region and Terminator region
Controls when, where and how much the gene is
expressed - viral promotors
Transit Peptide
Targets protein to correct organelle
Coding Region
Encodes protein product
vectors for transgenosis
Bacterial plasmids
Bacteriophage lambda (λ)
Cosmids - combination phages and plasmids
Integrated vectors
Artificial yeast chromosome, YAC
Viruses (adenoviruses, retroviruses, lentivirus) – mammals and
humans
Restriction endonuclease enzymes, which cut DNA
molecules at specific recognition nucleotide sequences
known as restriction sites palindroms
mostly bacterial enzymes
3. Insertion of vector to genome:
Transgenic mammals and gene therapy: introduction of
viral vectors, retroviruses, adenoviruses, lentiviruses with
recombinant DNA and transgene
Plant and bacterial transgenosis: introduction of
plasmids, phages, cosmids with recombinant DNA and
transgene by electric, heat or sound impulse, mikroinjection,
gene shooting, liposomes
4. selection of transformed organism and an
application of multiplied transgene or its protein in
a basic research of the gene and protein or
a medical, food or agricultural sciences and industry.
Transgenic plants with resistance and higher profitability.
Transgenic bacteria able to clean toxic wastes.
The application of protein generated by transgene for basic
research and for clinical use: insulin, human growth
hormon …
Application in Medicine
….also human albumin, monoclonal antibodies, antihemophilic factors, interferon, vaccines, enzymes.
Agriculture: production of Golden Rice
First…
Bacteria in 1973
Mice in 1974
Insulin-producing bacteria in 1982
Gene therapy in 1990
Genetically modified food has been sold since 1994
Trangenic animals
• are useful for research of gene therapy and human
pathologic genotypes and phenotypes
• expression of mammals genes
• model organism for testing vectors
Transgenic mice, sheeps, pigs, cattle…. hens, sheep, goats,
rabbits, pigs, cattle with resistance to diseases, to enhanced
quality of meat and milk
Methods important for medical sciences:
Production of monoclonal antibodies
VNTR variable number of tandem repeats
Single nucleotide polymorphisms SNPs
Allele specific oligonucleotide analysis (ASO)
Microarrays - Identifying sets of disease genes
Pharmacogenomics
Nanomedicine - may be used for delivery of small sensors to target
sites in body, detect and destroy cancer cells
Monoclonal antibodies
Microarrays
Dr. Peter Snustad, Michael J.Simmons:
Principles of Genetics, 5. edition, 2009
http://www.clinigene.eu/video-intro-gene-therapy.html
Thank you for your attention
Commercial cloning
Genetic donor
Somatic nucleus transfer
1 clon 32 000 USD
(r.2004)
Deposition of animal
cells into bank 850 USD
(r.2005)
clones
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