13-3 cell transformation
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This document discusses cell transformation, which is a change to a cell's DNA. It describes three methods of transforming cells: 1) Transforming bacteria cells using plasmids containing foreign DNA that can be inserted into the plasmid. The recombinant plasmids can then deliver the DNA into the bacterial cell. 2) Transforming plant cells either by using the bacterium Agrobacterium tumefaciens to transfer recombinant plasmids, or by using a gene gun to inject DNA-coated particles into plant cells. 3) Transforming animal cells by injecting foreign DNA directly into egg nuclei, or using gene replacement to "knock out" an existing gene.
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Cell transformation, by kk
INTRODUCTION
ROLE IN CELL LINE CHARACTERIZATION
CAUSES OF TRANSFORMATION
METHODS OF TRANSFECTION
CHARACTERISTICS OF TRAANSFORMED CELLS
GENETIC INSTABILITY
IMMORTALIZATION
ABRERANT GROWTH CONTROL
TUMORIGENECITY
CHROMOSOMAL ABERATION
APPLICATION
CONCLUSION
REFERENCE
GENE TRANSFER METHODS IN ANIMALS
Gene transfer methods in animals can be natural or artificial. Natural methods include conjugation, transformation, and transduction which transfer genes between bacteria. Artificial methods like microinjection, biolistics, liposome mediated transfer, calcium phosphate mediated transfer, and electroporation are used to directly insert genes into cells. These techniques transfer genes into organisms for genetic engineering applications such as producing transgenic animals, developing vaccines, and gene therapy to treat diseases.
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Transfection
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Primary culture and cell line
Introduction
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Steps In Primary Culture
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Dissection And/Or Disaggregation
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Mechanical Disaggregation
Cell Line( Finite & Continuous)
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Conclusion
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Biology and characterization of the cell culture
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Cell transformation, by kk
INTRODUCTION
ROLE IN CELL LINE CHARACTERIZATION
CAUSES OF TRANSFORMATION
METHODS OF TRANSFECTION
CHARACTERISTICS OF TRAANSFORMED CELLS
GENETIC INSTABILITY
IMMORTALIZATION
ABRERANT GROWTH CONTROL
TUMORIGENECITY
CHROMOSOMAL ABERATION
APPLICATION
CONCLUSION
REFERENCE
GENE TRANSFER METHODS IN ANIMALS
Gene transfer methods in animals can be natural or artificial. Natural methods include conjugation, transformation, and transduction which transfer genes between bacteria. Artificial methods like microinjection, biolistics, liposome mediated transfer, calcium phosphate mediated transfer, and electroporation are used to directly insert genes into cells. These techniques transfer genes into organisms for genetic engineering applications such as producing transgenic animals, developing vaccines, and gene therapy to treat diseases.
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Organ culture technique in synthetic media- animal tissue culture
Organ culture is a development from tissue culture that allows for the culture of pieces of organs on artificial media to accurately model organ functions in various states. Special culture methods are required as organs require high oxygen levels. Organ pieces can be cultured on plasma clots, agar, raft methods using lens paper or rayon, grid methods, or in liquid media using supports like gauze or rafts. Organ culture faces limitations as results may not match whole animal studies due to lack of in vivo drug metabolism.
Animal tissue culture
Cell culture involves removing cells from an animal or plant and growing them in an artificial environment that provides nutrients for growth. Key developments included the use of antibiotics to reduce contamination, enzymes like trypsin to detach adherent cells, and chemically defined media. Cell culture is used for modeling biology, toxicity testing, cancer research, virology, genetic engineering, and producing therapeutic proteins. Proper conditions like temperature, substrate, and media composition are required to keep cells "happy" and growing. Aseptic technique and containment are important to prevent contamination.
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In vitro testing of drug toxicity
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12. Cell synchronization and Immortalization
Cell synchronization helps in obtaining distinct sub population of cells representing different stages of cell cycle.It helps in collecting population wide data of cells progressing through various stages of cell cycle. Immortalization, refers to cells having capability of undergoing cell division infinitely. Immortal cells are particularly preferred in cell culture to enable long time storage and use. This presentation teaches about cell synchronization, methods of cell synchronization, cellular transformation, immortalization and mechanism of immortalization.
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SAGE (Serial Analysis of Gene Expression) is a technique that allows for the rapid and comprehensive analysis of gene expression patterns in a given cell population. It works by isolating mRNA, synthesizing cDNA, ligating short sequence tags to the cDNA, and then counting the number of times each tag is observed to quantify gene expression levels. The tags are concatenated and sequenced to generate vast amounts of data that must be analyzed computationally to identify which genes particular tags correspond to and to compare expression profiles between cell types. SAGE provides an overview of a cell's complete transcriptional activity and has been applied to study differences in cancer vs normal cells and to identify targets of oncogenes and tumor suppressor genes.
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Knockout mice are mice that have had a specific gene inactivated through replacement or disruption with artificial DNA. This allows researchers to study the function of that gene. The technique was awarded the 2007 Nobel Prize in Physiology. The procedure involves isolating the target gene, engineering a modified DNA sequence, introducing this into embryonic stem cells, and implanting the modified stem cells into mouse blastocysts. This generates chimeric mice that can pass the modified gene to offspring. Knockout mice provide insights into gene function in humans and are used as models for diseases. They also enable drug and therapy testing, though some genes cause developmental issues if knocked out.
Gene therapy and gene delivery systems
Genes carry hereditary information and encode proteins. Gene therapy aims to treat diseases by correcting defective genes through various approaches like inserting a normal gene or repairing an abnormal gene. Early research in the 1980s involved inserting human genes into bacteria. The first human gene therapy treated a girl for SCID in 1990 by inserting a normal gene into her white blood cells. Viral vectors like retroviruses and adenoviruses as well as non-viral methods can deliver genes, but all methods face challenges like short-lived effects, immune responses, and difficulties treating multi-gene disorders and diseases. While progress has been made, setbacks include a death in 1999 from an immune response and some children developing cancer from viral vectors.
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Mammalian cell culture, basic techniques
Introduction
Terminologies
Types of tissue culture
Applications
Culturing
Sub-culturing
Cryopreservation
Detection of contaminants
In vitro transformation of cells
Cell viability
Rules for working in the Lab
Advantages
Limitations
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Simply describe the yeast tow hybrid,where you can find definition ,principle,procedure,advantages and disadvantages .
Organ culture- animal tissue culture
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P br322
pBR322 is a 4,361 base pair plasmid vector originally constructed in 1977 for use in cloning experiments. It contains genes conferring resistance to ampicillin and tetracycline, which allow selection of recombinant clones, as well as an E. coli origin of replication. Recombinant selection involves insertional inactivation of the tetracycline resistance gene, rendering clones sensitive to tetracycline but resistant to ampicillin. pBR322 was widely used for cloning due to its small size, two selectable markers, and ability to be amplified in host cells. However, it is limited by its mobility between cells and small carrying capacity.
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This document discusses bioremediation and the use of microorganisms to degrade organic pollutants and remove contamination. It describes how bacteria, fungi and other microbes break down waste organic matter through metabolic processes. The document also discusses how genetic engineering can be used to design microorganisms capable of degrading specific contaminants more efficiently. Examples are provided of various bacteria and fungi that have been genetically modified or studied for their ability to break down pollutants like benzene, toluene, chlorobenzoate and heavy metals.
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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.
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Lipid mediated lipofection
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Chapter 4 isolation identification-and-cultivation
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2. Signs of viral growth include death, defects, or cytopathic effects in the host.
3. Infected tissues or fluids are examined microscopically or using techniques like PCR to identify the virus.
Cultivation of Viruses
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This document discusses various genetic engineering techniques including selective breeding, hybridization, inbreeding, and inducing mutations to increase genetic variation in organisms. It also describes manipulating DNA through techniques like extraction, cutting with restriction enzymes, gel electrophoresis, sequencing, PCR, and genetic engineering in bacteria, plants, and animals. Examples are given of applications of genetic engineering like producing insulin in bacteria and developing pest-resistant plants. Cloning is explained through the example of Dolly the sheep.
Transformation
This document describes the transformation of competent E. coli cells using the CaCl2 method. It defines competent cells as cells that can uptake exogenous DNA and discusses plasmids, which are self-replicating DNA that can carry useful genes. The document explains that transformation can be natural or artificial, and the artificial process involves making cells competent through ice-cold CaCl2 treatment, then applying heat shock to induce the cells to uptake exogenous DNA like plasmids.
Transformation in bacteria
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This document discusses recombinant DNA technology and its applications. It begins with an introduction to recombinant DNA technology and its history. It then describes the tools and enzymes used, including restriction enzymes, DNA ligase, reverse transcriptase, and DNA polymerase. Various vectors like bacterial plasmids and bacteriophages are also discussed. The document outlines several applications such as monoclonal antibody production, disease diagnosis, DNA fingerprinting, environmental uses, gene therapy, and xenotransplantation. In summary, the document provides an overview of the key concepts, techniques, and uses of recombinant DNA technology.
Glossary For Biotechnology
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Genetic engineering and recombinant DNA technology (1).pptx
Recombinant DNA technology involves combining DNA molecules from different sources that are not normally found together. It begins with restriction enzymes that cut DNA at specific sequences and DNA cloning vectors. DNA fragments are joined to the vector which is then placed into a host cell to replicate many copies. The copies are clones that can be studied. Recombinant DNA is used to isolate, replicate, and analyze genes. Key tools are restriction enzymes, plasmids, and genomic and cDNA libraries which allow screening for genes of interest.
29112019 Sahri Yanti DNA Recombinan Technology
DNA recombinant technology involves manipulating genes in organisms. It uses restriction enzymes to cut DNA at specific sites, vectors to carry foreign DNA, and host organisms for transformation. The key steps are:
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4) Selecting transformed host cells that contain the recombinant DNA
5) Analyzing the new genes and proteins produced by the host.
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Biotechnology uses organisms or biological systems to develop products and services. It has applications in agriculture, medicine, and industry. Recombinant DNA technology allows manipulation of genes between unrelated organisms. The process involves enzyme digestion of DNA, ligation of genes into vectors, introduction into host cells, and culturing to produce multiple copies of the gene. This technique has led to medical advances like insulin production and disease treatments.
Recombinant DNA technology AS PCI SYLLABUS
THE DNA RECOMBINANT TECHNOLOGY, THIS PRESENTATION INCLUDES THE GOALS OF THESE TECHNOLOGY, RDNA, ISOLATION OF DNA, CUTTING OF DNA, RESTRICTION ENZYMES AND ITS TYPES, AMPLIFICATION OF RECOMBINANT DNA, ENZYMES USED, HOST CELLS OF IR AND VECTORS USED; PLASMID VARIOUS VECTORS
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This document discusses two case studies involving genetically modified crops:
1) Drought tolerant transgenic plants developed using genes for abiotic stress tolerance through genetic engineering. Genes used include structural genes for late embryogenesis abundant (LEA) proteins and regulatory genes for key enzymes. This allows improved water stress management compared to conventional breeding.
2) Genetically engineered potatoes (Innate) modified using RNA interference to suppress expression of certain genes to reduce browning and increase resistance to bruising and soft rot. This helps improve potato quality and shelf life.
molecular biology
This document discusses rDNA technology and methods for gene cloning. It describes how recombinant DNA is formed by combining DNA sequences as desired. Gene cloning involves isolating a specific DNA fragment from an organism and introducing it into a plasmid vector that can replicate in a host cell, producing multiple copies of the fragment. Various methods are described for isolating DNA fragments, including mechanical shearing, restriction enzyme digestion, and reverse transcriptase. Different types of vectors like plasmids, bacteriophages, cosmids, BACs, and YACs that can be used are also summarized.
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Recombinant DNA technology uses restriction enzymes and DNA ligase to cut and join DNA fragments from different sources to construct recombinant DNA molecules. This technique was discovered in the 1970s and has since been used to develop transgenic plants with improved traits like higher yield, increased stress and pest resistance, and the ability to produce valuable pharmaceuticals. Some key applications include producing human insulin and anemia treatments, developing herbicide and insect resistant crop varieties, and engineering disease resistance in plants. Recombinant DNA technology is now widely used in agriculture and has contributed to over 70% of foods in supermarkets coming from genetically modified crops.
Recombinant dna technology jithin
Recombinant DNA technology involves joining DNA molecules from different species and inserting them into a host organism to produce new genetic combinations. This results in recombinant proteins being expressed within living cells. The process involves cloning the gene of interest and inserting it into a plasmid vector. This recombinant DNA is then transferred into a host cell, such as E. coli, and the transformed cells are selected and grown to produce the protein encoded by the inserted gene.
Recombinant DNA Technology
Now a day's these technique is tremendously use for in lab by using foreign Dna to to producing insulin in bacteria , plant with high yielding capacity by using Gene from another species
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Genetic engineering and Recombinant DNA
Genetic engineering involves altering the DNA of living organisms using biotechnology. It includes techniques like changing single DNA base pairs, deleting or adding genes, or combining DNA from different species. Recombinant DNA technology is used to create recombinant DNA molecules by manipulating DNA in vitro and introducing them into host organisms. This allows bacteria to be engineered to produce human insulin through inserting the human insulin gene into bacterial plasmids. Genomic libraries can be created by ligating fragmented genomic or cDNA into plasmid vectors to transform bacteria and clone the entire genome.
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DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid.- [https://www.khanacademy.org/science/...dna.../dna-cloning.../a/overview-dna-cloning]
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This document provides an overview of recombinant DNA technology. It discusses the basic principles, which involve generating DNA fragments, inserting a selected fragment into a cloning vector, introducing the vector into host cells, and multiplying the recombinant molecules. Key steps include using restriction enzymes to cut DNA at specific sites, ligases to join fragments, and various vectors like plasmids and bacteria to clone the DNA. The document also outlines several applications of rDNA technology, such as producing proteins, diagnosing diseases, and developing genetically engineered plants.
Genetic engineering
Genetic engineering involves the direct manipulation of genes through techniques like gene cloning and transfer. Key tools include restriction enzymes, plasmids, and the creation of DNA libraries which allow genes to be identified and isolated. Techniques like PCR, gel electrophoresis, and blotting allow analysis and study of DNA, RNA, and proteins. Restriction mapping uses restriction enzymes to cut DNA into fragments which can then be used to identify genes and construct maps of DNA sequences.
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Genetic engineering allows manipulation of a genome by introducing, eliminating, or modifying genes. The main steps are: 1) Isolating the gene of interest using restriction enzymes to cut DNA into fragments; 2) Inserting the gene into a bacterial plasmid using recombinant DNA techniques; 3) Detecting colonies containing the inserted gene through tests or hybridization; 4) Cloning the bacteria to produce many copies containing the gene.
Recombinant DNA Technology
Recombinant DNA technology allows DNA from different species to be isolated, cut, and spliced together to form new recombinant molecules. Key tools for recombinant DNA technology include restriction enzymes, ligases, polymerases, vectors, and host cells. Recombinant DNA technology has many applications, including producing human insulin and other proteins for medical use, genetically engineering plants for crop improvement, and DNA fingerprinting for criminal investigations.
Genetic engineering and recombinant DNA technology
Genetic Engineering is a set of techniques capable to allow the identification, manipulation and multiplication of genes of living organisms.
E-CONTENT BIOTECH-1
Recombinant DNA technology involves combining DNA molecules from different sources into new combinations that would not otherwise be found in nature. This is done through several steps: isolating the desired DNA fragment, cutting it with restriction enzymes, joining it to a vector molecule like a plasmid, and inserting the new recombinant DNA into a host cell where it can be replicated in large quantities. Applications of this technology include producing human insulin in bacteria for medical use and mapping genomes to study genetic disorders and biological processes. However, some concerns have also been raised about potential negative environmental and safety impacts.
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13 4 applications of genetic engineering
Genetic engineering can be used to modify cells and organisms by transferring genes. This includes creating transgenic bacteria, animals, and plants by introducing foreign DNA. Examples given are plants that produce pesticides, cotton resistant to herbicides, tobacco that produces human proteins, and mustard plants that create bioplastic. The first genetically modified primate, a monkey named ANDi, was also introduced. Dolly the sheep was the first mammal cloned from an adult cell nucleus, implanted into a surrogate mother. Ethical issues around who decides what is "corrected" and modifying animals for human organ transplants were also raised.
13-2 manipulating DNA
The document describes the basic techniques of genetic engineering which include extracting DNA from cells, cutting DNA into fragments using restriction enzymes, separating DNA fragments through gel electrophoresis, amplifying DNA through polymerase chain reaction, and making recombinant DNA by cutting and recombining DNA from different species and inserting it into host cells. These techniques allow scientists to isolate, modify, and transfer genes between organisms.
12-4
This document discusses different types of mutations including gene mutations and chromosomal mutations. It describes point mutations such as single base substitutions, insertions, and deletions as well as frameshift mutations that can occur from insertions or deletions. It also discusses chromosomal mutations like duplications, inversions, translocations, and deletions. Non-disjunction during meiosis can result in aneuploidy with an extra or missing chromosome. Mutations can be harmful and cause genetic disorders or cancer, but some are beneficial and lead to evolution or traits in polyploid plants. Examples of genetic disorders and polyploid crops are also provided.
12-3
The DNA base code that codes for the amino acid recognized by the tRNA with the anticodon UAC would be TAC. This is because:
- The tRNA anticodon UAC pairs with the mRNA codon AUG
- In DNA, T pairs with A and A pairs with U
- Therefore, the complementary DNA sequence that would code for the mRNA codon AUG is TAC.
So the DNA base code for the amino acid recognized by the tRNA with anticodon UAC is TAC.
12-2
The document summarizes DNA replication in prokaryotic and eukaryotic cells. In prokaryotes, DNA replication begins at a single point and proceeds bidirectionally along the circular chromosome. In eukaryotes, replication begins at many sites and also proceeds bidirectionally along the linear chromosomes, which are packaged into nucleosomes and further condensed into chromatin. The semi-conservative model of replication is described, in which the parental DNA strands separate and each serves as a template for a new complementary strand.
12 1
Griffith's transformation experiments showed that a component from dead S strain bacteria could transform live R strain bacteria to the S strain, causing disease. Avery later showed that the transforming factor was DNA. Hershey and Chase's experiment, using radioactive labeling of bacteriophage DNA and protein, demonstrated that DNA, not protein, entered the bacterial cell and was responsible for heredity transfer. Together, these experiments provided strong evidence that DNA is the genetic material.
11_4
Meiosis produces gametes through two rounds of cell division beginning with one diploid cell and ending with four haploid cells. In meiosis I, homologous chromosomes pair and separate, reducing the chromosome number by half. Meiosis II separates sister chromatids, further dividing the cell and its genetic material. The steps of crossing over and independent assortment in meiosis I contribute to genetic variation among gametes and offspring.
11 3
This document discusses several genetic inheritance patterns beyond simple dominance and recessiveness:
1) Codominance - Both alleles contribute to the phenotype, such as a cow that is both red and white producing a roan coat color.
2) Multiple alleles - A single gene can have more than two alleles influencing a trait, such as four alleles determining rabbit coat color.
3) Sex linkage - Some traits are determined by genes on the X chromosome, making them more common in one sex, like color blindness being more prevalent in males.
11 1 2012-h
DNA is copied when mitosis or meiosis begins, forming new cells with identical or varying chromosomes. Gregor Mendel conducted experiments with pea plants in the mid-1800s and discovered that traits are inherited as discrete units called factors, now known as genes, which segregate independently during gamete formation according to his laws of inheritance. His work established the foundations of classical genetics.
11 5 linkage
1) Gene linkage refers to genes that are located on the same chromosome and are usually inherited together into the same gametes. It is the chromosomes, not individual genes, that assort independently.
2) Linked genes may separate into different gametes during crossing over, which occurs more frequently between genes that are farther apart on the same chromosome.
3) A gene map shows the relative locations of known genes on a chromosome but not their exact positions.
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13-3 cell transformation
- 1. 13-3 Cell Transformation (Changes in Cell’s DNA)
- 2. Review • Which enzyme is used as a scissor in genetic engineering ? Which one is used as glue? Sciessor: Restriction Enzyme Glue: DNA ligase • What’s the function of gel electrophoresis? Separate DNA fragments by their different size
- 3. 13-3 Cell transformation • Cell transformation= change the cell’s DNA • A cell takes DNA from outside. The external DNA becomes part of the cell’s DNA
- 4. 1) Transforming bacteria cells – Using plasmid • small circle of extra bacterial DNA, double strand • Foreign DNA can be inserted into plasmid – Forms recombinant plasmids – Plasmids used in genetic engineering are called vectors. – Can deliver DNA into another cell
- 5. Plasmid
- 6. 1) Transforming bacteria cells – Using plasmid • Plasmid has a origin site for replication • Plasmid has a genetic marker (usually a gene for resistance to antibiotics) • two types of plasmid integration into a host bacteria
- 7. Restriction enzyme DNA or cDNA Foreign DNA, plasmid cuts molecule fragments with DNA, and modification of chromosomal DNA sticky ends enzymes are mixed or cDNA recombinant plasmids containing foreign DNA Same enzyme cuts plasmid DNA same sequence in with sticky ends plasmid DNA host cells containing recombinant plasmids
- 8. What if insertion eukaryotic DNA into bacteria ? Will there be any problems ???
- 9. Transcription RNA polymerase mRNA splicing DNA Pre-RNA mRNA No introns Bacteria don’t know how to deal with introns USE cDNA!!!
- 10. Reverse transcription= Making cDNA mRNA transcript Reverse transcriptase cDNA -- A DNA reverse transcription from a mRNA–cDNA hybrid matured mRNA ` “c” -- complementary single-stranded cDNA DNA polymerase double-stranded cDNA
- 12. 2) Transforming plant cells • Recombinant plasmid is transformed to plant – By using bacterium Agropbacterium tumefaciens – By gene gun injection
- 13. By using Agrobacterium Plasmids carrying the gene of interest is transferred to agrobacterium The agrobacterium inserts the foreign DNA into the plant cell to form transgenic cell
- 14. By using gene gun To coat metal particles with gene containing plasmids Gene gun injects the DNA covered particles into the plant cell
- 19. 3) Transforming animal cells • Animal cell – Inject into egg nucleus some enzymes may help to insert foreign DNA into chromosomes – Gene replacement ( gene “knockout”)
Editor's Notes
- Knock out particular genes