Biotechnology Gene therapy, Cloning Heredity No 439 by RNDr. Hana Zoubkov

1
BiotechnologyGene therapy, CloningHeredity No
439by RNDr. Hana Zoubková, PhD
2
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
3
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
4
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

5
(No Transcript)
6
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
infants teeth Stem cells may be obtained by
reprogramming somatic cells
7
Sources of adult and infant stem cells
8
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
9
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.
10
1
2
4
3
11
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) patients
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.
12
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 In vivo GAT using recombinant adenoviruses or liposomes to deliver the CFTR gene
Familial hypercholesterolemia Liver cells Ex vivo GAT using recombinant retroviruses to deliver the LDL receptor gene (LDLR)
Gaucher's disease glucocerebrosidase Hemopoietic stem cells Ex vivo GAT using retroviruses to deliver the gene (GBA)
13
Severe combined immune deficiency
Mutation in gene encoding adenine deaminase enzyme
Shows up as incompetent T-cells which prevent
B-cells from making antibodies
14
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
15
Molecular cloning the principle of bacterial
production of insulin, it is used a vector with
transgene (insulin gene)
16
1. Identification, isolating unknown gene by
Homology cloning based on a similarity to known
genes. e.g. Mouses 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
17
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.
18
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 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
19
vectors for transgenosis
Bacterial plasmids Bacteriophage lambda (?)
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
20
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
21
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
22
(No Transcript)
23
Application in Medicine .also human albumin,
monoclonal antibodies, anti-hemophilic 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
24
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

25
Methods important for medical sciences Productio
n 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
26
Monoclonal antibodies
27
Microarrays
28
Dr. Peter Snustad, Michael J.Simmons Principles
of Genetics, 5. edition, 2009
http//www.clinigene.eu/video-intro-gene-therapy.h
tml
Thank you for your attention
29
(No Transcript)
30
Commercial cloning

Somatic nucleus transfer 1 clon 32 000 USD
(r.2004) Deposition of animal cells into bank
850 USD (r.2005)