This document provides an overview of protein expression in mammalian cells and protein purification techniques. It discusses how mammalian cells are the preferred system for producing mammalian proteins due to their ability to perform post-translational modifications. Common mammalian cell lines for expression include HEK293 and CHO cells. Plasmid vectors are typically used to insert the gene of interest, and transfection methods like lipofection and electroporation can be used to introduce the plasmid into cells. Affinity, size-exclusion, and ion-exchange chromatography are common protein purification techniques.
S1 Mapping is a laboratory method used for locating the start and end points of
transcripts and for mapping introns.
This technique is used for quantifying the amount of mRNA transcripts, it can therefore identify the level of transcription of the gene in the cell at a given time.
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
This document discusses the technique of DNA shuffling. It begins with a brief history of DNA shuffling, invented by Willem P.C. Stemmer in 1994. It then discusses the goal of DNA shuffling, which is to generate libraries of chimeric variants from related DNA sequences that can undergo directed evolution. Critical parameters for DNA shuffling include using a thermostable DNA polymerase and performing a fragmentation step using MnCl2 instead of MgCl2. Applications of DNA shuffling discussed include evolving improved enzymes, cytokines, antibodies, viral proteins, and vaccine candidates.
Vectors for gene transfer in animals: Retro virusKhushbu
Retroviruses are RNA viruses that can be used as vectors to transfer genes into host cells. The retroviral genome consists of two RNA strands that are reverse transcribed into DNA and integrated into the host cell chromosome. This integrated viral DNA, or provirus, can then be used to express the transferred gene. Retroviruses are useful for gene transfer because the introduced gene is stably integrated and replicated in daughter cells, but they require cell division for integration and may cause insertional mutagenesis. The document discusses the basic structure and life cycle of retroviruses, constructs used to make replication-defective retroviral vectors, and the steps involved in gene transfer using retroviruses.
This document discusses key components of expression vectors that are important for efficiently expressing cloned genes. It explains that expression vectors contain regulatory sequences like promoters and terminators to control transcription, as well as elements like ribosome binding sites, fusion tags, and selection markers. Specifically, it provides details on tightly regulated promoters, commonly used viral and bacterial promoters, and considerations for promoters in prokaryotic and eukaryotic expression systems. The document also reviews other important vector elements and their functions.
A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.
This document discusses the yeast two-hybrid system, which is used to detect protein-protein interactions. It describes the original yeast two-hybrid system developed by Fields and Song, as well as several variations that can detect other types of molecular interactions. These variations are referred to as "n-hybrid systems". The document then provides details on the principle and setup of the classic yeast two-hybrid system, as well as alternative yeast two-hybrid systems that detect interactions in different cellular locations.
Lectut btn-202-ppt-l23. labeling techniques for nucleic acidsRishabh Jain
Nucleic acid probes can be labeled with radioisotopes or nonisotopic labels for use in hybridization techniques. Common labeling methods include radioactive labeling with 32P or 3H, or nonisotopic labeling with biotin, digoxigenin, or fluorescein. Labeled probes are used to detect complementary DNA or RNA sequences and can be DNA, RNA, or oligonucleotide probes. Probes are prepared through various techniques such as PCR, random priming, or in vitro transcription and must be purified before use and stored appropriately.
S1 Mapping is a laboratory method used for locating the start and end points of
transcripts and for mapping introns.
This technique is used for quantifying the amount of mRNA transcripts, it can therefore identify the level of transcription of the gene in the cell at a given time.
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
This document discusses the technique of DNA shuffling. It begins with a brief history of DNA shuffling, invented by Willem P.C. Stemmer in 1994. It then discusses the goal of DNA shuffling, which is to generate libraries of chimeric variants from related DNA sequences that can undergo directed evolution. Critical parameters for DNA shuffling include using a thermostable DNA polymerase and performing a fragmentation step using MnCl2 instead of MgCl2. Applications of DNA shuffling discussed include evolving improved enzymes, cytokines, antibodies, viral proteins, and vaccine candidates.
Vectors for gene transfer in animals: Retro virusKhushbu
Retroviruses are RNA viruses that can be used as vectors to transfer genes into host cells. The retroviral genome consists of two RNA strands that are reverse transcribed into DNA and integrated into the host cell chromosome. This integrated viral DNA, or provirus, can then be used to express the transferred gene. Retroviruses are useful for gene transfer because the introduced gene is stably integrated and replicated in daughter cells, but they require cell division for integration and may cause insertional mutagenesis. The document discusses the basic structure and life cycle of retroviruses, constructs used to make replication-defective retroviral vectors, and the steps involved in gene transfer using retroviruses.
This document discusses key components of expression vectors that are important for efficiently expressing cloned genes. It explains that expression vectors contain regulatory sequences like promoters and terminators to control transcription, as well as elements like ribosome binding sites, fusion tags, and selection markers. Specifically, it provides details on tightly regulated promoters, commonly used viral and bacterial promoters, and considerations for promoters in prokaryotic and eukaryotic expression systems. The document also reviews other important vector elements and their functions.
A DNA library is a collection of DNA fragments that have been cloned into vectors. DNA libraries allow researchers to isolate and study specific DNA fragments of interest. To create a genomic library, DNA is extracted from an organism, cut into fragments, inserted into vectors, and introduced into host bacteria to generate clones containing all the organism's DNA sequences. This library can then be screened to identify and study particular genes. DNA libraries provide an efficient way to store, isolate, and analyze DNA sequences.
This document discusses the yeast two-hybrid system, which is used to detect protein-protein interactions. It describes the original yeast two-hybrid system developed by Fields and Song, as well as several variations that can detect other types of molecular interactions. These variations are referred to as "n-hybrid systems". The document then provides details on the principle and setup of the classic yeast two-hybrid system, as well as alternative yeast two-hybrid systems that detect interactions in different cellular locations.
Lectut btn-202-ppt-l23. labeling techniques for nucleic acidsRishabh Jain
Nucleic acid probes can be labeled with radioisotopes or nonisotopic labels for use in hybridization techniques. Common labeling methods include radioactive labeling with 32P or 3H, or nonisotopic labeling with biotin, digoxigenin, or fluorescein. Labeled probes are used to detect complementary DNA or RNA sequences and can be DNA, RNA, or oligonucleotide probes. Probes are prepared through various techniques such as PCR, random priming, or in vitro transcription and must be purified before use and stored appropriately.
pUC vectors are plasmids derived from pBR322 that have a higher copy number of 500-600 copies per cell. They contain an ampicillin resistance gene for selection, as well as the lacZ' gene containing multiple cloning sites. When a gene of interest is inserted, it disrupts the lacZ' gene, allowing for blue-white screening on media containing IPTG and X-gal to identify recombinant colonies that appear white instead of blue. pUC vectors offer advantages over pBR322 such as high copy number and easy selection, though they cannot accommodate inserts larger than 15kb.
This document discusses various types of vectors used for cloning, including bacteriophage vectors, plasmid vectors, cosmid vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and shuttle vectors. Key points include:
- Bacteriophage derivatives like lambda phage are suitable for cloning large eukaryotic DNA due to abilities like packaging millions of clones and size selection of DNA.
- Phage-based vectors can be insertional, containing a single cloning site, or replacement vectors with two cloning sites allowing DNA substitution.
- Cosmids are hybrid phage-plasmid vectors that can package DNA up to 48 kb into phage particles.
- Y
This document discusses the history and properties of plasmids. Plasmids are extrachromosomal DNA molecules that are able to replicate independently of a cell's chromosomal DNA. They were first observed in bacteria in the early 1950s and play important roles in processes like antibiotic resistance and virulence. The document outlines the early studies that helped characterize plasmids and describes some of their key properties, such as their circular structure and ability to exist in different conformations. It also discusses how plasmids are used as cloning vectors to amplify genes and produce proteins for applications in research, medicine, and agriculture.
This document discusses adenoviral cloning vectors. It begins by defining a cloning vector as a small piece of DNA that can be stably maintained in an organism and have foreign DNA inserted into it for cloning purposes. It then discusses viral vectors, noting that they are commonly used to deliver genetic material into cells through transduction. The document focuses on properties of viral vectors, specifically safety features and targeting abilities. It provides details on adenoviruses, noting they can efficiently transfer genes, their structure, applications in gene therapy and vaccination, and their DNA genome capacity. Adeno-associated viruses are also mentioned as attractive for gene therapy due to mild immune response.
Manipulation of gene expression in prokaryotesSabahat Ali
For expression of gene in a particular vector, always used strong regulatable promoter (lac promoter, trp promoter, tac promoter , trc promoter, pL promoter, T7 gene promoter)
use of dual plasmid system & fusion proteins
How we can increase our protein product yield?
This document discusses different expression systems for producing recombinant proteins, including prokaryotic, yeast, insect cell, and mammalian systems. It provides details on some commonly used expression vectors such as pGEX-3X plasmid for prokaryotic expression in E. coli, Saccharomyces cerevisiae and Pichia pastoris yeast expression systems using episomal and integrating plasmids, and baculovirus expression in insect cells using the polyhedrin promoter to drive expression of the gene of interest. The key advantages and limitations of different expression systems are also summarized.
PGEM3-Z is a plasmid vector derived from pUC vectors. It contains the ampicillin resistance gene (ampR) and LacZ gene with clustered restriction sites, similar to pUC vectors. PGEM3-Z is commonly used for in vitro transcription, protein expression, and DNA cloning due to its high copy number, ability to identify inserts through blue-white screening, and flexibility afforded by clustered restriction sites.
A comprehensive study of shuttle vector & binary vector and its rules of in ...PRABAL SINGH
Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
- Baculovirus expression vectors allow genes of interest to be expressed in insect cells. The gene is inserted into the baculovirus genome, downstream of the strong polyhedrin promoter.
- When the recombinant baculovirus infects insect cells, the gene is highly expressed. This system is useful for producing recombinant proteins.
- T7 expression systems also allow high level expression of genes in bacteria. The gene of interest is placed in a plasmid downstream of the T7 promoter. This plasmid is introduced into E. coli containing the T7 RNA polymerase gene, which is induced to express the polymerase.
The document discusses the plasmid vector pBR322, which was constructed in 1977 and is one of the most commonly used cloning vectors. It describes the origins and components of pBR322, including two antibiotic resistance genes, the origin of replication, and restriction enzyme cleavage sites. The document also summarizes the construction of several derivatives of pBR322, including pBR327, pUC18, and pBR118/119, and notes their applications and advantages over the original pBR322 vector.
This document discusses commonly used protein expression systems. It begins by explaining how recombinant DNA technology has enabled the cloning and expression of mammalian genes in different systems to produce therapeutic and vaccine proteins. It notes that the suitable expression system depends on factors like productivity, bioactivity, purpose and protein characteristics. The document then examines various prokaryotic (bacterial) and eukaryotic expression systems in detail, including E. coli, Bacillus subtilis, Lactococcus lactis, Pseudomonas, Corynebacterium, yeast, mammalian cells and baculovirus cells. It highlights advantages and disadvantages of each system for recombinant protein expression.
Artificial chromosomes can be constructed in vitro from defined DNA components to carry large DNA fragments stably like natural chromosomes. There are different types of artificial chromosomes including BACs, YACs, PACs, and HACs. BACs can carry up to 300 kbp of DNA and are useful for genome sequencing. YACs allow cloning of up to 500 kbp of foreign DNA in yeast cells but clones can be unstable. PACs derive from bacteriophage P1 and carry 100-300 kbp of DNA. HACs are human-sized artificial chromosomes that can act as new chromosomes in human cells, carrying therapeutic genes.
Gene cloning involves making exact copies of a particular gene or DNA sequence using genetic engineering techniques. The key steps are:
1. Isolating the gene of interest from an organism's DNA.
2. Inserting the isolated gene fragment into a vector, such as a plasmid, to form recombinant DNA.
3. Introducing the recombinant DNA into a host cell, usually a bacterium, where it can replicate. As the host cells divide, numerous identical copies of the gene are produced.
Gene cloning is used to isolate and amplify genes, determine nucleotide sequences, identify control sequences and mutations, and engineer organisms for applications like producing insulin or making crops insect-resistant.
This document discusses different types of DNA libraries and methods for screening libraries to identify clones containing genes of interest. It describes genomic and cDNA libraries, noting that genomic libraries contain all DNA fragments from an organism's genome while cDNA libraries contain only coding sequences. The key screening methods discussed are colony/plaque hybridization using radiolabeled probes, expression screening using antibodies, and PCR screening using gene-specific primers.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
Expression of recombinant proteins in mammalian cell linesSandeep Kumar
The speaker discusses mammalian cell-based recombinant protein production. Mammalian cells like CHO cells are commonly used as they can properly fold and modify proteins, similar to human cells. Issues include mammalian cells being fragile, slow-growing, and techniques being expensive. Benefits are low immunogenicity and high safety due to not being susceptible to human pathogens.
Chromosome walking is a method used to isolate and clone a particular gene or allele through positional cloning. It involves using overlapping clones that contain DNA fragments near the target gene to "walk" through the chromosome until reaching the gene. Each successive clone is tested to map its precise location until eventually reaching the target gene. Chromosome walking was developed in the early 1980s and can be used to analyze genetically transmitted diseases and find single nucleotide polymorphisms. However, it has limitations such as being a slow process and difficulty walking through repeated sequences.
The document discusses the pET plasmid expression system used for recombinant protein expression in E. coli. It notes that the pET system uses strong T7 promoters to drive high expression of cloned genes, but this could be toxic to host cells. The pET plasmid contains the gene for T7 RNA polymerase under control of the lac promoter and lac repressor. In the presence of IPTG, T7 RNA polymerase is expressed and binds the T7 promoter on the plasmid to transcribe the cloned gene. This allows for tightly regulated, high-level expression of recombinant proteins without overloading the host cell.
Nucleotide excision repair (NER) removes DNA damage caused by ultraviolet light that was discovered in 1964. NER can be divided into two pathways: global genomic NER that repairs the entire genome and transcription coupled NER that repairs the template strand of actively transcribed genes. Xeroderma pigmentosum is a rare genetic disorder where NER is deficient, causing extreme sun sensitivity and increased risk of skin cancer. Cockayne syndrome is a also rare but fatal neurodegenerative disorder where cells cannot preferentially repair UV damage in transcribed genes.
The document discusses various methods of transfection in animals. Transfection is the process of introducing nucleic acids into eukaryotic cells. It describes viral transfection using bacteria like Agrobacterium tumefaciens and viruses. Non-viral methods include chemical transfection using calcium phosphate, liposomes, polyamines. Mechanical transfection employs microinjection or particle bombardment. Common chemical methods are calcium phosphate precipitation, polyplexes, and liposomes/lipoplexes. Viruses used are retroviruses, adenoviruses, adeno-associated viruses. Bacterial and viral vectors allow for integration into the host genome while chemical and mechanical are often transient.
This document discusses various gene transfer techniques including physical, chemical, and biological methods. It focuses on biological methods such as bactofection and transduction using viruses. Bactofection involves using bacteria to deliver genes directly into cells, while transduction uses viruses to package and deliver genes. The document also discusses chemical methods like calcium phosphate and lipofection, as well as physical methods such as electroporation, microinjection, and particle bombardment to introduce DNA into host cells.
pUC vectors are plasmids derived from pBR322 that have a higher copy number of 500-600 copies per cell. They contain an ampicillin resistance gene for selection, as well as the lacZ' gene containing multiple cloning sites. When a gene of interest is inserted, it disrupts the lacZ' gene, allowing for blue-white screening on media containing IPTG and X-gal to identify recombinant colonies that appear white instead of blue. pUC vectors offer advantages over pBR322 such as high copy number and easy selection, though they cannot accommodate inserts larger than 15kb.
This document discusses various types of vectors used for cloning, including bacteriophage vectors, plasmid vectors, cosmid vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and shuttle vectors. Key points include:
- Bacteriophage derivatives like lambda phage are suitable for cloning large eukaryotic DNA due to abilities like packaging millions of clones and size selection of DNA.
- Phage-based vectors can be insertional, containing a single cloning site, or replacement vectors with two cloning sites allowing DNA substitution.
- Cosmids are hybrid phage-plasmid vectors that can package DNA up to 48 kb into phage particles.
- Y
This document discusses the history and properties of plasmids. Plasmids are extrachromosomal DNA molecules that are able to replicate independently of a cell's chromosomal DNA. They were first observed in bacteria in the early 1950s and play important roles in processes like antibiotic resistance and virulence. The document outlines the early studies that helped characterize plasmids and describes some of their key properties, such as their circular structure and ability to exist in different conformations. It also discusses how plasmids are used as cloning vectors to amplify genes and produce proteins for applications in research, medicine, and agriculture.
This document discusses adenoviral cloning vectors. It begins by defining a cloning vector as a small piece of DNA that can be stably maintained in an organism and have foreign DNA inserted into it for cloning purposes. It then discusses viral vectors, noting that they are commonly used to deliver genetic material into cells through transduction. The document focuses on properties of viral vectors, specifically safety features and targeting abilities. It provides details on adenoviruses, noting they can efficiently transfer genes, their structure, applications in gene therapy and vaccination, and their DNA genome capacity. Adeno-associated viruses are also mentioned as attractive for gene therapy due to mild immune response.
Manipulation of gene expression in prokaryotesSabahat Ali
For expression of gene in a particular vector, always used strong regulatable promoter (lac promoter, trp promoter, tac promoter , trc promoter, pL promoter, T7 gene promoter)
use of dual plasmid system & fusion proteins
How we can increase our protein product yield?
This document discusses different expression systems for producing recombinant proteins, including prokaryotic, yeast, insect cell, and mammalian systems. It provides details on some commonly used expression vectors such as pGEX-3X plasmid for prokaryotic expression in E. coli, Saccharomyces cerevisiae and Pichia pastoris yeast expression systems using episomal and integrating plasmids, and baculovirus expression in insect cells using the polyhedrin promoter to drive expression of the gene of interest. The key advantages and limitations of different expression systems are also summarized.
PGEM3-Z is a plasmid vector derived from pUC vectors. It contains the ampicillin resistance gene (ampR) and LacZ gene with clustered restriction sites, similar to pUC vectors. PGEM3-Z is commonly used for in vitro transcription, protein expression, and DNA cloning due to its high copy number, ability to identify inserts through blue-white screening, and flexibility afforded by clustered restriction sites.
A comprehensive study of shuttle vector & binary vector and its rules of in ...PRABAL SINGH
Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
- Baculovirus expression vectors allow genes of interest to be expressed in insect cells. The gene is inserted into the baculovirus genome, downstream of the strong polyhedrin promoter.
- When the recombinant baculovirus infects insect cells, the gene is highly expressed. This system is useful for producing recombinant proteins.
- T7 expression systems also allow high level expression of genes in bacteria. The gene of interest is placed in a plasmid downstream of the T7 promoter. This plasmid is introduced into E. coli containing the T7 RNA polymerase gene, which is induced to express the polymerase.
The document discusses the plasmid vector pBR322, which was constructed in 1977 and is one of the most commonly used cloning vectors. It describes the origins and components of pBR322, including two antibiotic resistance genes, the origin of replication, and restriction enzyme cleavage sites. The document also summarizes the construction of several derivatives of pBR322, including pBR327, pUC18, and pBR118/119, and notes their applications and advantages over the original pBR322 vector.
This document discusses commonly used protein expression systems. It begins by explaining how recombinant DNA technology has enabled the cloning and expression of mammalian genes in different systems to produce therapeutic and vaccine proteins. It notes that the suitable expression system depends on factors like productivity, bioactivity, purpose and protein characteristics. The document then examines various prokaryotic (bacterial) and eukaryotic expression systems in detail, including E. coli, Bacillus subtilis, Lactococcus lactis, Pseudomonas, Corynebacterium, yeast, mammalian cells and baculovirus cells. It highlights advantages and disadvantages of each system for recombinant protein expression.
Artificial chromosomes can be constructed in vitro from defined DNA components to carry large DNA fragments stably like natural chromosomes. There are different types of artificial chromosomes including BACs, YACs, PACs, and HACs. BACs can carry up to 300 kbp of DNA and are useful for genome sequencing. YACs allow cloning of up to 500 kbp of foreign DNA in yeast cells but clones can be unstable. PACs derive from bacteriophage P1 and carry 100-300 kbp of DNA. HACs are human-sized artificial chromosomes that can act as new chromosomes in human cells, carrying therapeutic genes.
Gene cloning involves making exact copies of a particular gene or DNA sequence using genetic engineering techniques. The key steps are:
1. Isolating the gene of interest from an organism's DNA.
2. Inserting the isolated gene fragment into a vector, such as a plasmid, to form recombinant DNA.
3. Introducing the recombinant DNA into a host cell, usually a bacterium, where it can replicate. As the host cells divide, numerous identical copies of the gene are produced.
Gene cloning is used to isolate and amplify genes, determine nucleotide sequences, identify control sequences and mutations, and engineer organisms for applications like producing insulin or making crops insect-resistant.
This document discusses different types of DNA libraries and methods for screening libraries to identify clones containing genes of interest. It describes genomic and cDNA libraries, noting that genomic libraries contain all DNA fragments from an organism's genome while cDNA libraries contain only coding sequences. The key screening methods discussed are colony/plaque hybridization using radiolabeled probes, expression screening using antibodies, and PCR screening using gene-specific primers.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
Expression of recombinant proteins in mammalian cell linesSandeep Kumar
The speaker discusses mammalian cell-based recombinant protein production. Mammalian cells like CHO cells are commonly used as they can properly fold and modify proteins, similar to human cells. Issues include mammalian cells being fragile, slow-growing, and techniques being expensive. Benefits are low immunogenicity and high safety due to not being susceptible to human pathogens.
Chromosome walking is a method used to isolate and clone a particular gene or allele through positional cloning. It involves using overlapping clones that contain DNA fragments near the target gene to "walk" through the chromosome until reaching the gene. Each successive clone is tested to map its precise location until eventually reaching the target gene. Chromosome walking was developed in the early 1980s and can be used to analyze genetically transmitted diseases and find single nucleotide polymorphisms. However, it has limitations such as being a slow process and difficulty walking through repeated sequences.
The document discusses the pET plasmid expression system used for recombinant protein expression in E. coli. It notes that the pET system uses strong T7 promoters to drive high expression of cloned genes, but this could be toxic to host cells. The pET plasmid contains the gene for T7 RNA polymerase under control of the lac promoter and lac repressor. In the presence of IPTG, T7 RNA polymerase is expressed and binds the T7 promoter on the plasmid to transcribe the cloned gene. This allows for tightly regulated, high-level expression of recombinant proteins without overloading the host cell.
Nucleotide excision repair (NER) removes DNA damage caused by ultraviolet light that was discovered in 1964. NER can be divided into two pathways: global genomic NER that repairs the entire genome and transcription coupled NER that repairs the template strand of actively transcribed genes. Xeroderma pigmentosum is a rare genetic disorder where NER is deficient, causing extreme sun sensitivity and increased risk of skin cancer. Cockayne syndrome is a also rare but fatal neurodegenerative disorder where cells cannot preferentially repair UV damage in transcribed genes.
The document discusses various methods of transfection in animals. Transfection is the process of introducing nucleic acids into eukaryotic cells. It describes viral transfection using bacteria like Agrobacterium tumefaciens and viruses. Non-viral methods include chemical transfection using calcium phosphate, liposomes, polyamines. Mechanical transfection employs microinjection or particle bombardment. Common chemical methods are calcium phosphate precipitation, polyplexes, and liposomes/lipoplexes. Viruses used are retroviruses, adenoviruses, adeno-associated viruses. Bacterial and viral vectors allow for integration into the host genome while chemical and mechanical are often transient.
This document discusses various gene transfer techniques including physical, chemical, and biological methods. It focuses on biological methods such as bactofection and transduction using viruses. Bactofection involves using bacteria to deliver genes directly into cells, while transduction uses viruses to package and deliver genes. The document also discusses chemical methods like calcium phosphate and lipofection, as well as physical methods such as electroporation, microinjection, and particle bombardment to introduce DNA into host cells.
Hi, I am RAFi ,student of Genetic Engineering and Biotechnology , Jashore university of science & Technology. It is my first uploading slide in slideshare.I am so glad for doing this work.
This document discusses methods for transferring genes between organisms through chemical means. It describes calcium phosphate and lipid-mediated gene transfer, which are two common chemical methods. Calcium phosphate forms a precipitate that binds DNA and transfers it into cells, while lipid-mediated transfer uses liposomes to encapsulate DNA and fuse with cell membranes to deliver the DNA. Both methods can be used for transient or stable transfection but have limitations such as low efficiency. The document also provides details on the mechanisms, advantages, and applications of these chemical gene transfer methods.
Transfection in animal cells through chemical methods like Calcium phosphate ...SmritiRanjan9
The document summarizes various methods for transfecting animal cells, including calcium phosphate coprecipitation, lipofection, electroporation, viral vectors, microinjection, and direct DNA transfer. It provides details on the mechanisms and advantages/disadvantages of each method. Calcium phosphate transfection involves precipitating DNA with calcium phosphate to facilitate cell binding and entry. Lipofection uses cationic lipids to form complexes with nucleic acids for cellular uptake. Electroporation exposes cells to electric pulses to temporarily destabilize membranes and allow DNA entry. Viral vectors are highly efficient but require biosafety precautions. Microinjection and gene guns directly inject or bombard DNA into cells.
This document discusses various methods of transfection, which is defined as the introduction of foreign DNA into eukaryotic cells. It describes transfection methods such as calcium phosphate transfection, liposome-mediated transfection, retroviral transfection, and electroporation. It provides details on how each method works and compares their strengths and weaknesses. Common transfection methods like calcium phosphate and liposomes are simple but have low efficiency, while retroviral transfection can generate stable cell lines but has limitations on DNA size. Electroporation is fast and applicable to many cell types.
Gene cloning involves producing exact copies of a gene using genetic engineering techniques. It involves isolating the gene of interest from one organism and inserting it into a vector, which is then introduced into a host organism where the gene can be replicated. There are several methods used to transfer genes between organisms or cells, including bacterial transformation, electroporation, transfection, and microinjection. Bacterial transformation involves directly taking up exogenous DNA, electroporation uses an electric pulse to create pores for DNA entry, while transfection introduces nucleic acids into eukaryotic cells using chemical reagents or viruses.
Transfection is a technique used to insert foreign nucleic acids like DNA or RNA into cells to alter their properties. There are various biological, chemical, and physical methods to accomplish transfection, either transiently or stably. In stable transfection, the foreign DNA integrates into the cellular genome and is passed to daughter cells, while transient transfection only expresses the DNA for a short time without integration. Genetic engineering techniques are used to transfer genes between organisms, like retrovirus-mediated gene transfer where the retrovirus acts as a vector to deliver transgenes into host cells. Embryonic stem cell-mediated gene transfer involves introducing DNA into stem cells that can integrate randomly or through homologous recombination and be passed to offspring. Liposome
DNA Transfection in Animal tissue culture and its methods.pptxMethusharma
You will learn the definition of DNA transfection in this presentation its examples, along with the procedures that are employed, through the use of organised flowcharts and diagrams. The Animal Biotechnology course, it is the first technique to learn.
Electroporation uses electric pulses to create temporary pores in the cell membrane, allowing DNA entry. DNA-coated microprojectiles are accelerated into cells using a gene gun. Microinjection precisely inserts DNA into cells through fine glass needles. Calcium phosphate precipitation forms DNA-calcium phosphate complexes taken up by cells. Cationic liposomes fuse with cell membranes, transferring DNA across. Adenoviruses and retroviruses can deliver DNA to dividing and non-dividing cells. Agrobacterium transfers tumor-inducing (T-DNA) from its Ti plasmid into plant cells at wound sites.
BOC Sciences is a life science group with its headquarter in New York. We are dedicated to providing the most complete in vivo and in vitro nucleic acid and protein transfection solutions to support gene and cell therapy, biologics production, and life science research.
Genetic transformation method in mammals cell by NIDHI MISHRA and tahura mari...Tahura Mariyam Ansari
this presentation includes method of gene transfer, factor that affect efficiency of gene transfer, fate of DNA in the recipient cells, autonomous replication vector and some other subtopics.
Genetic transformation & success of DNA ligation Sabahat Ali
DNA is ligated through DNA Ligase, problems may occur during DNA ligation are
1) vector cyclization
2) vector-vector concatemers
3) target DNA-target DNA ligation
This document discusses non-viral gene transfer methods. It describes various techniques for direct delivery of naked DNA including electroporation, gene guns, sonoporation, magnetofection, hydrodynamic delivery, and microinjections. It also discusses various non-viral vectors for gene delivery including oligonucleotides, liposomes, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides. Each method is described in terms of its mechanism of delivery, advantages, disadvantages and suitable target tissues. The document provides an overview of non-viral gene expression systems and delivery methods.
Gene delivery is the process of introducing foreign DNA into host cells. There are four principal mechanisms for transferring genes into animal cells: direct physical transfer through microinjection or particle bombardment; chemical-mediated transfection using calcium phosphate or liposomes; transduction using viral vectors; and bactofection using bacterial vectors. Mammalian cells are widely used hosts as they allow production of recombinant human proteins with authentic post-translational modifications. Important applications include gene therapy and producing therapeutic proteins.
This document discusses various methods for transferring genes into animal cells, including viral and non-viral approaches. Viral methods use viruses like adenovirus to transfer genes, while non-viral methods include biochemical techniques like calcium phosphate transfection, lipid-mediated transfection using lipofectamine, and physical methods like microinjection, particle bombardment/gene guns, ultrasound, and electroporation. The document provides detailed protocols for lipid-mediated transfection and some of the other non-viral methods.
This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate transfection, liposome-mediated transfection, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, how transformation involves direct DNA uptake by competent bacteria, and how transduction involves transfer of DNA between bacteria via bacteriophages. The document also discusses Agrobacterium-mediated plant transformation and applications of gene transfer techniques.
This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate and liposome mediated transfer, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, and how electroporation uses electrical pulses to create pores in cell membranes to allow DNA entry. The document also summarizes screening and applications of transgenic techniques.
Microinjection is a gene transfer technique where DNA is directly injected into cells using a fine glass micropipette. It is highly efficient at the individual cell level and was originally used for transfecting hard-to-transfect cells. The procedure involves holding a cell using one pipette while another pipette is used to inject DNA into the cell's cytoplasm or nucleus. It allows for stable transfection efficiencies of around 20% and is used to generate transgenic animals by injecting DNA into oocytes, eggs or embryos. However, it is time-consuming and can only be done for a small number of cells.
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2. A mammalian host system is the preferred expression
platform for producing mammalian proteins that have the
most native structure and activity.
Mammalian expression is the system of choice for
studying the function of a particular protein in the most
physiologically relevant environment, because it allows for
the highest level of posttranslational processing and
functional activity of the protein.
They have been successfully applied in the
biopharmaceutical production of cytokines, monoclonal
antibodies, growth factors and so on.
For mammalian cell expression , the gene of interest
insert through the vector based method and transfection
methods.
PROTEIN EXPRESSION IN MAMMALIAN
CELLS
3. 2 TYPES OF MAMMALIAN CELLS COMMONLY
USED
HEK293 (Human
Embryonal Kidney 293)
Cells
• Human Embryonic Kidney
293 (HEK293) cells are a
specific cell line originally
derived from human
embryonic kidney cells
grown in tissue culture.
• They are very easy to grow
and transfect very readily,
thus widely used in cell
biology research and
biotechnology industry.
• Transient expression
CHO (Chinese Hamster
Ovary) cells
• Chinese hamster ovary
(CHO) cells are a cell line
derived from the ovary of
the chinese hamster.
• They’re often used in
biological and medical
research.
• Transient and stable
expressions
4. 2 TYPES OF CELL EXPRESSION
Inducible
Expression
• An inducible promoter allows controlling the timing of
gene expression. In the absence of the inducer, the
gene is not expressed.
• This option is ideal for expressing toxic proteins.
• For regulated and inducible expression of the gene of
interest, use the Invitrogen T-REx Expression System,
Invitrogen Flp-In T-REx system.
Constitutive
Expression
• if working with a nontoxic gene and the timing of
expression is not important, choose an expression
vector with a constitutively expressing promoter.
• For this purpose, use the pcDNA vectors.
• pcDNA vectors are available with either the CMV,
EF-1, or UbC promoter, a variety of different epitope
tags, standardized detection or purification of
proteins, several selection markers, and different
cloning formats.
5. PLASMID BASED EXPRESSION
VECTOR
Expression vector is a vector that allows the
transcription and translation of a foreign gene inserted
into it.
Eukaryotic expression vector is a shuttle vector, containing ColE1 ori,
SV40 origin and has an efficient promoter (e.g. SV40 early promoter,
Rous Sarcoma virus promoter, adenovirus major late promoter, or
human cytomegalovirus promoter).
In most cases, first attempts to transiently express recombinant
proteins were executed with ‘standard’ expression vectors that
contain strong viral promoters, such as SV40 or a CMV promoter
from cytomegalovirus (CMV).
More recently, at least one non-viral promoter, the
elongation factor (EF-1) promoter. It is appears to be
as strong or stronger than some viral promoters.
6.
7. TRANSFECTION
Transfection is defined as the process of inserting nucleic
acids (e.g., plasmid DNA, cDNA, mRNA, miRNA, siRNA)
into the eukaryotic cells. In addition, proteins and
nanoparticles such as beads or dyes can be transfected.
Transfection is an important and widely used tool for
analyzing the function of various genes and used for
gene silencing via RNAi, gene editing via CRISPR/Cas9,
as well as over expression studies by cellular integration
of plasmid DNA, mRNA, or proteins.
Infection:- viral-mediated process in which target cells are
infected with a virus carrying cloned DNA sequence in its
genome
8. TYPE OF TRANSFECTION
STABLE
• Viral and Microinjuction
based
• Long term Expression
• Foreign gene non-
specific integrated into
genome
• Complex process
TRANSIENT
• Chemical and
Electroporation based
• Short Term Expression
(24-96hr)
• Foreign gene not
integrated into genome
• Easy process
11. PHYSICAL METHODS
The target cell is positioned under a microscope
and being fixed by a pipette then the nucleotide
solution is then directly injected into the
cytoplasm and/or the nucleus using a fine glass
capillary needle.
However, as each single cell needs to be
microinjected individually, this method is very time-
consuming, expensive but highly effective.
Microinjection is mostly used for single cell
manipulation or the generation of transgenic animals.
1.
Microinjuction
12.
13. During electroporation, a mixture of the cells and the
nucleotide of interest is exposed to an intense electric field.
This leads to transient cell membrane destabilization,
making the cell membrane permeable to the nucleotides that
are present in the surrounding solution, to enter the
cytoplasm.
After removing the electric field, the cell membrane stabilizes,
enclosing the nucleotides in the cytoplasm, where they are
expressed.
Electroporation allows for the transient and stable
transfection of any cell type.
This method is easy and reliable, but it requires high cell
numbers due to high rates of cell death during the procedure.
Therefore, electroporation is not suitable for sensitive and
difficult-to-culture cell types, such as primary cells.
2. Electroporation
14.
15. CHEMICAL METHODS
A mixture of neutral and cationic liposomes forms complexes with the
nucleotides of interest. These complexes pass the cell membrane and
then release the nucleotides into the cytoplasm via endocytosis. Then,
escape the endosome or undergo lysosomal degradation. After
successful endosomal escape, the nucleotide is expressed in the
target cells.
Lipofection is commonly used to transfer nucleic acids such as RNA or DNA into
eukaryotic cells.
This method is easy to apply and yields highly reproducible results of transient and
stable transfections.
However, lipofection efficiency strongly depends on the cell type and has to be
tested and optimized in advance.
Especially for primary and non-dividing cells, the viability after the transfection
process might be decreased due to the high cellular sensitivity.
1.
Lipofe
ction
16.
17. 2. CALCIUM PHOSPHATE
A mixture of the nucleotides, calcium, and phosphate buffer
forms a precipitate that is taken up by the cells via
endocytosis. Then, the nucleotides either escape the
endosome or undergo lysosomal degradation.
The calcium phosphate transfection is an insoluble precipitate
with DNA and for both transient and stable transfection. It is an
inexpensive and simple method for transient or stable
nucleotide transfer into most cell lines.
The transfection efficiency, strongly depends on the cell
constitution, the pH, and the quality and the amount of the
used nucleotides.
The calcium phosphate transfection is toxic and therefore not
suitable for most sensitive primary cell lines.
18.
19. 3. CATIONIC POLYMERS
The negatively charged nucleotide backbones form complexes
with cationic polymers, such as diethylaminoethyl (DEAE)-
dextran. The complex is then taken up by the cells, mostly via
endocytosis. If no lysosomal degradation occurs, the nucleotide
can escape from the endosome into the cytosol of the host cells,
subsequently resulting in transgene expression.
Using cationic polymers, nucleotides can be transiently
transfected into eukaryotic cells in an inexpensive and simple
manner.
As a drawback, transfection by cationic polymers has a very low
efficiency (<10%) in a number of cell types, including primary
cells.
Further, cationic polymers are highly cytotoxic and therefore not
suitable for transfection of sensitive cells and generation of stable
cell lines.
20.
21. VIRUS BASED MAMMALIAN
TRANSFECTION
Adenoviruses are a class of double stranded DNA viruses that efficiently
deliver nucleotides directly into target cells. The virus enters the host cell
via endocytosis. Following endosomal escape, the viral genome is
transported into the nucleus, where it is expressed by the replication
machineries of the host cell.
Nowadays, replication-deficient adenoviruses are widely used for
transduction and gene therapy, due to their high efficiency and low
pathogenicity.
Adenoviral vectors have proven to be a very successful transduction tool
in many eukaryotic cell types, such as human and rodent cells. Besides
dividing cell lines, this method gives access to difficult-to-transfect cells,
such as primary cells.
Adenovirus-mediated transduction is always transient, meaning that no
nucleotide integration into the host genome occurs. Transduction
efficiencies of up to 100% can easily be achieved.
1. Adenoviral
Transduction
22.
23. 2. LENTIVIRAL
TRANSDUCTION
Lentiviruses a subclass of retroviruses have the ability to
permanently integrate into the genome of the host cell. After
the virus has entered the cell, the viral RNA is transcribed by
the reverse transcriptase to produce double-stranded DNA
that enters the nucleus. Finally, the transgene is integrated
into the host genome via the lentiviral integrase enzymes.
Lentiviruses enable stable gene transfer in vitro and in vivo, as
they integrate into the host cell genome and offer the
possibility of positive cell selection.
They have a broad host cell range allowing transduction of
many cell types such as primary neurons, lymphocytes, and
macrophages. Moreover, lentiviral vectors have also proven to
be effective in transducing brain, liver, muscle, and retina in
vivo without toxicity or immune responses.
24.
25. COMMONLY USED TECHNIQUES FOR
PROTEIN PURIFICATION
1. Affinity
Chromatography
2. Gel
permeation
3. Ion Exchange
Chromatography
The most common methods for preparative
purification of proteins all involve chromatography.
26. 1. AFFINITY
CHROMATOGRAPHY
Affinity Chromatography is a separation technique based upon
molecular conformation.
A ligand which can make a complex with specific protein (dextran,
polyacrylamide, cellulose etc) binds the filling material of the
column through the spacer arm.
The specific protein which makes a complex with the ligand is
attached to the solid support (matrix), and retained in the column,
while free proteins leave the column.
The target protein leaves the column by means of changing its
ionic strength through alteration of pH or addition of a salt solution.
This chromatography technique is used for the purification of
enzymes, hormones, antibodies, nucleic acids, and specific
proteins.
27.
28. 2. GEL- PERMEATION (MOLECULAR SIEVE)
CHROMATOGRAPHY
The basic principle of this method is to use dextran containing materials to
separate macromolecules based on their differences in molecular sizes.
In a gel- permeation column stationary phase consists of inert molecules with
small pores. The solution containing molecules of different dimensions are
passed continuously with a constant flow rate through the column.
Smaller Molecules cannot permeate into gel particles, and they are retained
between particles within a restricted area while Larger molecules pass through
spaces between porous particles, and move rapidly through inside the
column. As a result, Firstly larger molecule elute out from column, then the
retained smaller molecules eluted later.
This procedure is basically used to determine molecular weights of proteins,
and to decrease salt concentration of protein solutions.
Sephadex-G type is the most frequently used column material. Besides,
dextran, agorose, polyacrylamide are also used as column materials.
29.
30. 3. ION- EXCHANGE
CHROMATOGRAPHY
Ion- exchange chromatography is based on electrostatic
interactions between charged protein groups, and solid support
material (matrix).
Matrix has an ion load opposite to that of the protein to be
separated, and the affinity of the protein to the column is
achieved with ionic ties.
Proteins are separated from the column either by changing pH,
concentration of ion salts or ionic strength of the buffer solution.
Positively charged ion- exchange matrices are called anion-
exchange matrices, and adsorb negatively charged proteins.
While matrices bound with negatively charged groups are
known as cation-exchange matrices, and adsorb positively
charged proteins.
31.
32. REFERENCES
•Felgner P.L., et al. Lipofection: a highly efficient, lipid-mediated DNA-
transfection procedure. 1987, Proc Natl Acad Sci U S A,
10.1073/pnas.84.21.7413.
•Lee C.S., et al. Adenovirus-mediated gene delivery: Potential
applications for gene and cell-based therapies in the new era of
personalized medicine. Genes Dis, 2017,
10.1016/J.GENDIS.2017.04.001.
•Sakuma T., Barry M.A., Ikeda Y. Lentiviral vectors: basic to translational.
Biochem J, 2012, 10.1042/BJ20120146.
•Meissner P, Pick H, Kulangara A, et al. Transient gene expression:
recombinant protein production with suspension-adapted HEK293-EBNA
cells[J]. Biotechnol Bioeng, 2001, 75(2): 197-203.
•Cockett M I, Bebbington C R, Yarranton G T. High level expression of
tissue inhibitor of metalloproteinases in Chinese hamster ovary cells
using glutamine synthetase gene amplification[J]. Biotechnology (N Y),