What is Cell-Cell Hybridization?
History
More about Somatic cell Hybridization
Mapping of genes by somatic cell Hybridization
Hybridoma technology
Other Applications of Somatic Cell Hybridization
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
cell lineage , cell fate - diverse class of cell fate, cell fate in plant meristem, mammalian development cell fate, nutritional effects on epigenetics, epigenetics of plants,
control of cell fate.
A knockout mouse is a mouse in which a specific gene has been inactivated or“knocked out” by replacing it or disrupting it with an artificial piece of DNA.
The loss of gene activity often causes changes in a mouse's phenotype and thus provides valuable information on the function of the gene.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
The SPECIAL - GIANT CHROMOSOMES which are very transcriptionally active DNA, where loops of DNA emerging from an apparently continuous chromosomal axis are coated with RNA polymerase.
Comparatively much larger than polytene chromosomes.
Highly significant for scientific analysis especially regarding gene amplification.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Introduction
Genetics of somatic cell
Somatic cell genetics
Somatic cell nuclear transfer
Somatic cell hybridization
Mapping human genes by using human rodent hybrids
In medical application
Production of monoclonal antibodies by using hybridoma technology
Conclusion
References
cell lineage , cell fate - diverse class of cell fate, cell fate in plant meristem, mammalian development cell fate, nutritional effects on epigenetics, epigenetics of plants,
control of cell fate.
A knockout mouse is a mouse in which a specific gene has been inactivated or“knocked out” by replacing it or disrupting it with an artificial piece of DNA.
The loss of gene activity often causes changes in a mouse's phenotype and thus provides valuable information on the function of the gene.
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
The SPECIAL - GIANT CHROMOSOMES which are very transcriptionally active DNA, where loops of DNA emerging from an apparently continuous chromosomal axis are coated with RNA polymerase.
Comparatively much larger than polytene chromosomes.
Highly significant for scientific analysis especially regarding gene amplification.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Introduction
Genetics of somatic cell
Somatic cell genetics
Somatic cell nuclear transfer
Somatic cell hybridization
Mapping human genes by using human rodent hybrids
In medical application
Production of monoclonal antibodies by using hybridoma technology
Conclusion
References
Students of medical and allied subjects must be exposed to the concept of monoclonal antibodies for the efficient practice of clinical and laboratory medicine.
Introduction
Genetics of somatic cell
Somatic cell genetics
Somatic cell nuclear transfer
Somatic cell hybridization
Mapping human genes by using human rodent hybrids
In medical application
Production of monoclonal antibodies by using hybridoma technology
Conclusion
References
BIOTECHNOLOGY IS
CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ......
ITS A VERY INTERESTING TO LEARN ABOUT HYBRIDOMA TECHNOLOGY .. THEIR PRODUCTION AND
APPLICATION ALSO ....
Hybridoma
Hybridomas are cells that have been engineered to produce a desired antibody in large amounts, to produce monoclonal antibodies.
Monoclonal antibodies can be produced in specialized cells through a technique now popularly known as hybridoma technology.
Hybridoma technology was discovered in 1975 by two scientists, G. Kohler and C. Milstein, were awarded Noble prize for physiology and medicine in 1984.
Facts about DNA
Eukaryotic chromosomes
Chemical composition of eukaryotic chromosomes
Histones
Non-histone chromosomal protein
Scaffold proteins
Folded fibre model
Nucleosome model
H1 proteins
Histone modification
Chromatosome
Higher order of chromatin structure
Mechanism of DNA packaging
Conclusion
Introduction
2. Thermoregulation
3. Vant Hoff equation
4. Temperature effect on cells
5. Extreme cold : resistance and death
6. Extreme heat : resistance and lethal death
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
2. CONTENTS
What is Cell- Cell Hybridization?
History
More about Somatic cell Hybridization
Mapping of genes by somatic cell Hybridization
Hybridoma technology
Other Applications of somatic Cell Hybridization
Conclusion
Reference
3. WHAT IS CELL- CELL
HYBRIDIZATION?
– CELL CELL HYBRIDIZATION:
– The technique of hybrid production through the fusion of somatic
cells under in vitro conditions, is called Somatic Hybridization
– Somatic Cell Hybridization involves the fusion of two different cells
to produce a hybrid which is termed as Heterokaryon
– The product of fusion was called Homokaryon if the two parental
cells come from the same species
4.
5. Creation of a PC-12 homokaryon. The cells were originally located at a distance of about 100 μm
from each other. By using the microelectrodes, they were placed next to each other. The cells were
pretreated with di-electrophoresis . A shows cells immediately after di-electrophoresis, and a close
contact is visualized by the flattened contact zone between the cells.
6. HISTORY
• In vitro somatic cell hybridization was first discovered by George Barski in
Paris in 1960
• Barski reported that cultures containing a mixture of two sublines of
heteroploidy mouse cell were overgrown by new cell lines, the karyotype of
which was the sum of two parental karyotypes
• Lederberg(1958) and Pontecorvo in 1961 sensed the potential of this new
approach.
Ephrussi confirmed the validity of the observation by Barski’s Group
7. MORE ABOUT SOMATIC
HYBRIDIZATION
-Somatic cell hybridization are of two types
• Spontaneous Hybridization is very rare
• -Induced Hybridization is mediated by either chemically with Polyethylene
Glycol, which effects the cell membrane, or with inactivated virus for
example the Sendai Virus.
SPONTANEOUS INDUCED
8. MORE ABOUT SOMATIC CELL
HYBRIDIZATION
• Hybridization can either be performed between two same species
which is termed as interspecific
• Or it can also be performed between two different species
Intraspecific
9. SOMATIC HYBRIDIZATION IN
ANIMAL CELL
– Somatic cells of different types can be fused to obtain hybrid
cells. Hybrid cells are useful in a variety of ways, e.g.,
– (i) for gene or chromosome mapping
– (ii) production of monoclonal antibodies by
producing hybridoma
10. GENE MAPPING BY SOMATIC
CELL HYBRIDIZATION
– The ability to distinguish each human chromosome is required to perform somatic-cell
hybridization, in which human and mouse (or hamster) cells are fused in culture to form a
hybrid.
– The fusion is usually mediated chemically with polyethylene glycol, which affects cell
membranes; or with an inactivated virus, for example the Sendai virus, that is able to fuse
to more than one cell at the same time.
– When two cells fuse, their nuclei are at first separate, forming a heterokaryon, a cell with
nuclei from different sources.
11. MAPPING OF GENES BY SOMATIC
CELL HYBRIDIZATION
CELL FUSION
SELECTION OF HYBRIDS
CHROMOSOME LOSS
CHROMOSOMAL MAPPING
12. CELL FUSION
– The commonly used fusion agents for mapping of chromosome are Sendai virus,
lysolecithin, liposomes and polyethylene glycol (PEG).
– The most widely used fusion agent is Sendai virus, which is inactivated by UV light or
β-propiolactone
– The process of fusion by Sendai virus involves 3 stages –
The virus particles cause cell agglutination
Cytoplasmic bridges are formed between cells
Cytoplasmic bridges expand to form spherical fused cells
15. SELECTION OF HYBRIDS
– To isolate pure population of Human –Mouse Heterokaryon a selection procedure is used
that kills both the parental cells and the homokaryons but allows the human – mouse
hybrid cells to survive and grow.
– In the medium contains a drug aminopterin, which blocks the de novo purine and
pyrimidine biosynthetic pathways of cells.
– However , the presence of hypoxanthine and thymidine the cells can overcome the block
by synthesizing their purines and pyrimidines using salvage pathways
16.
17. SELECTION OF HYBRIDS
– For cells to grow in HAT medium, two enzymes, hypoxanthine-guanine
phosphoribosyl transferase (HGPRT) and thymidine kinase (TK) must be functional.
– Mouse cell is deficient in TK (TK-) and a human cell is deficient in HGPRT(HGPRT-)
– Heterokaryons , has normal HGPRT gene derived from the mouse genome and TK
from gene from the human genome. Therefore only hybrid cell grows.
18. CHROMOSOME LOSS IN
HYBRID CELLS
– When hybrids are formed between rodent and human cells , human
chromosomes are lost preferentially.
– Loss of chromosome in interspecific hybrids was noted in fusion between
mouse and rat cells., where chromosomes of rat were preferentially lost.
– In rodent and human cell fusions, up to 95% of the human chromosomes
can be lost by the time the hybrids are initially isolated and their
chromosome analyzed.
19.
20. MAPPING STRATEGIES
– The qualities of rodent –human hybrids that make useful for
human mapping are-
– Preferential loss of human chromosomes (in most cases)
– Easy detection or unilateral loss of human phenotypes and
differentiation from their rodent counterpart
21. SYNTENY TESTING
– This is based on the premise that genes located on the same chromosome
will be retained and lost concordantly from hybrid cells
– Gene pairs or groups were determined to be syntenic by observing the
pattern of retention and loss in a large number of independently derived
hybrid cell line.
– However this must be supported by chromosomal analysis to rule out loss of
a pair of chromosomes or extensive chromosome rearrangements that would
obscure a proper syntenic relationship
22. •Product A can be assigned to chromosome 5.
• Product B can be assigned to chromosome 3.
• Product C is not on any of the chromosomes 1-7.
• Product D can be assigned to chromosome 1.
23. ASSIGNMENT BY SELECTION
– The first assignment of genes to chromosomes using rodent-human hybrids
relied on the ability to select for the genes.
– The assignment of the TK gene to the chromosome 17 was facilitated by
isolation of reduced which contained only chromosome 17 in common.
– TK- mouse cells were fused with human cells in HAT medium and the hybrid
cells were examined, and only chromosome 17 was found to be common,
which would be carrying the TK gene
24.
25. BASIC HYBRIDOMA
TECHNOLOGY
– Hybridomas are cells that have been engineered to produce a desired antibody in
large amounts, to produce monoclonal antibodies.
– Monoclonal antibodies can be produced in specialized cells through a technique now
popularly known as hybridoma technology.
– Hybridoma technology was discovered in 1975 by two scientists, Georges Kohler of
West Germany and Cesar Milstein of Argentina .
26.
27. STAGES
– The method developed by Kohler and Milstein involves four stages which
results in the production of hybrid lymphocyte with an infinite growth
capacity and capable of continuous of a single antibody.
– The stages of this process are shown:
Immunization
Cell fusion
Genetic Selection
Cell selection
28. IMMUNIZATION
– The first stage of production of a hybridoma is to obtain lymphocytes
from and animal that is enriched with specific antibody- secreting
cells.
– Antibodies are synthesized by B lymphocytes which can be isolated
from the spleen of an immunized animal.
29. METHODS OF FUSION:-
– Cells can be induced to fuse if two cell populations are brought close together at
a high cell concentration in the presence of viruses or by chemical agents (called
‘fusogens’).
– The process involves the destabilization of adjacent cell membranes which
eventually fuse to form a hybrid cell.
– Although UV-inactivated Sendai viruses were originally used as agents for cell
fusion, the more widely used method is now fusion by the chemical agent
polyethylene glycol (PEG). This is a polymer, available at a molecular weight
range of 200–20000 kDa.
30.
31. SELECTABLE GENE MARKERS
FOR CELL SELECTION
– The process of cell fusion will result in a heterogeneous population
of cells that will contain unfused parental cells, lysed cells as well
as the required hybrid cells. At this stage, cell selection is
important so that the hybrid cells can be isolated from the
mixture. For hybridomas there are two important stages of cell
selection:
■ isolation of hybrid cells from parental cells;
■ selection of antibody-secreting cells within the hybrid cell
population.
32. Clonal selection of Mab-secreting
hybridomas
• After genetic selection with HAT the culture contains hybridomas but only some of
these will secrete antibodies.
• The next stage involves selection of Mab-secreting hybridomas from the
population which has survived HAT treatment Cell clones can be isolated by the
method of limiting dilution.
• Cloning ensures that all cells selected for future cultures are genetically identical.
33. OTHER APPLICATIONS OF
SOMATIC CELL HYBRIDIZATION
– 1. Somatic hybridization has helped to study the cytoplasmic genes and their functions.
In fact, the information is successfully used in plant breeding programmes.
– 2. Protoplast fusion will help in the combination of mitochondria and chloroplasts to
result in a unique nuclear-cytoplasmic genetic combination.
– 3. Somatic hybridization can be done in plants that are still in juvenile phase.
– 4. Protoplast transformation (with traits like nitrogen fixation by incorporating
exogenous DNA) followed by somatic hybridization will yield innovative plants.
34. CONCLUSION
– Somatic Cell Hybridization is the process of fusion which takes place between
two Somatic Cell.
– Somatic cell hybridization is usually done between mouse and human cells, but
many other intraspecific or interspecific hybridizations can be done.
– It is widely used for the production of Monoclonal Antibodies and for the Gene
Mapping of Human Chromosomes.
– Even though this presentation specifies on animal cell, this process can be used
for plant cell hybridization.
35. REFERENCE
- Brackett B. J., New technologies in animal breeding, Elsevier 2012 pg- 171- 193
– Jaffe E. A., Biology of Endothelial cells, Springer Science and Business Media,2012. Pg- 194
– Lodish H, Berk A, Zipursky SL, et al., 2000. Molecular Cell Biology, 4th edition, W. H. Freeman.
– Ephrussi B., Hybridization of Somatic Cells, Princeton University Press 2015. pg-166
– Crow J. F. and Dove W. F., 2000. Perspectives on Genetics: Anecdotal, Historical, and Critical Commentaries
1987-1998, Univ of Wisconsin Press
– McConkey E H,, 1993. Human Genetics: The Molecular Revolution, Jones & Bartlett Learning
– Yunis J., 2012. Molecular Structure of Human Chromosomes, Elsevier.
– Meurant G., (1977). Methods in Cell Biology, Volume 15, Academic Press