Exam 3 Terms

methylase

-DNA -an enzyme that adds methyl groups to adenine bases in bacterial DNA and cytosine in eukaryotic DNA; in bacteria the addition of a methyl group on a base of the recognition site of a restriction endonuclease prevents the enzyme from cleaving the bacterial DNA

luciferase

-Luc -from firefly -enzyme used in pyrosequencing to generate light for detection of individual nucleotides added to a nascent DNA, one at a time -oxidizes luciferin, yielding a fluorescent product that can be quantified by measuring the released light

differentiated cell

-a cell specialized towards specific functions -less than 10% of the all the genes in the genome are expressed at high levels, and a large fraction of genes are silenced

cDNA library

-a collection of DNA sequences generated from mRNA expressed in a particular cell or tissue type at a given time -this type of library contains only protein-coding DNA sequences and does not include any noncoding DNA (such as regulatory elements or introns)

insert

-a foreign DNA molecule -can be joined to a vector in a ligation reaction

knockout mouse

-a genetically engineered mouse in which researchers have inactivated an existing gene by replacing it or disrupting it with an artificial piece of DNA by gene targeting -"loss-of-function" mutants These are often created by gene targeting so scientists can study the knockout mouse as a model for that particular disease

DNA probes

-a key element required to identify a gene during library screening -cloned DNA -can be ss or ds, but only useful as probes when ss and therefore must be denatured before use -DNA that has the same or a similar sequence to that of a specific gene or DNA sequence of interest, such that the denatured probe and target DNA can hybridize when they are renatured together -a nucleic acid probe can detect a complementary molecule in a complex mixture with exquisite sensitivity and specificity

plasmid

-a small, double-stranded circular (or linear) DNA molecule carried by a bacteria, some fungi, and some higher plants -naturally occurring -extrachromosomal, independent, and self-replicating

cosmid

-a vector carrying a phage lambda cos site, allowing it to be packaged into a phage head -infect a host bacterium as do phages, but they replicate like plasmids and the host cells are not lysed

cohesive sites/cohesive ends

-aka cos sites -short regions of single-stranded DNA whose base sequences are complementary to each other at each end of a linear genome -sticky cohesive ends: formed by a staggered cut by a restriction enzyme - the ends can hydrogen bond to the single-stranded complementary tails of other DNA fragments

ddNTP

-aka replication terminator -a synthetic nucleotide that lacks a 3' OH group, and is thus unable to form a 3'-5' phosphodiester bond necessary for chain elongation; used in enzymatic DNA sequencing

restriction enzyme

-aka restriction endonuclease -restrict or prevent viral infection by degrading the invading nucleic acid -an enzyme that recognizes specific base sequences of double-stranded DNA and cuts the DNA at or near these sites

fluorophore

-an intrinsic peptide -a group of atoms in a molecule responsible for absorbing light energy and producing the color of the compound

BAC

-bacterial artificial chromosome -a vector based on the E.coli F plasmid capable of holding foreign DNA inserts up to 300,000 base pairs -once the foreign DNA has been cloned and transformed into the host bacteria, many copies of it can be made

phage

-bacteriophage -a virus that infects bacteria (literally "bacterium eater")

recombination

-breaking and rejoining (of a genome) -reassortment of genes or alleles in new combinations -occurs by crossing-over within or between DNAs

vector

-can replicate independent of host genome replication in host cells -an agent, such as a virus or a plasmid, phage, or artificial chromosome, that carries a modified or foreign gene -used as a carrier in gene cloning experiments. When used in gene therapy, a vector delivers the desired gene to a target cell

CAT

-chloramphenicol acetyltransferase -enzyme encoded by a bacterial gene that adds acetyl groups to the antibiotic chloramphenicol -frequently used as a reporter gene in eukaryotic transcription/translation experiments

cDNA

-complementary DNA -a DNA copy of an RNA, made by reverse transcription

ELISA

-enzyme-linked immunosorbent assay -an immunoassay used to quantify antigen-antibody reactions by combining the specificity of antibodies with the sensitivity of simple enzyme assays

blunt ends

-formed by restriction enzymes that cut both strands of DNA at the same position -no unpaired nucleotides

GFP

-green fluorescent protein -from jellyfish -commonly used as a reporter gene -naturally fluorescent, doesn't require substrates or cofactors for light production

multiple cloning site

-has a number of unique target sites for restriction endonucleases -when cut here, the result is a linear plasmid

in situ

-hybridization -the base pairing of a labeled probe to metaphase chromosomes on a microscope slide, or to RNA to determine the precise localization within a cell

DNA ligases

-join two pieces of DNA end-to-end by forming phosphodiester bonds

LB-SMGT

-linker-based sperm-mediated gene transfer -a method for making transgenic animals in which the foreign DNA is bound to sperm via a monoclonal antibody; the sperm is then used to fertilize an egg

expression library

-made with a cloning vector that contains the required regulatory elements for gene expression -useful for identifying a clone containing the cDNA of interest when an antibody to the protein encoded by that gene or cDNA is available

PCR

-polymerase chain reaction -a target sequence of DNA can be amplified a billionfold in several hours -procedure is carried out entirely in vitro

southern blotting

-transferring DNA fragments separated by gel electrophoresis to a support medium such as nitrocellulose or nylon membrane, in preparation for hybridization to a labeled probe -used to identify and locate DNA sequences which are complementary to the probe Primary purpose is to identify a specific gene fragment from the often many DNA bands on a gel.

YAC

-yeast artificial chromosome -extremely large segments of DNA from another species spliced into artificial chromosomes of yeast -YACS are used to clone up to one million bases of foreign DNA into a host cell, where the DNA is propagated along with the host cell chromosomes

• Be able to explain the steps involved in cloning an animal using nuclear transfer.

1. Preparation of donor cells.- source of donor cells must either be totipotent embryonic stem cells or differentiated somatic cells. The donor nucleus and the egg must be in the same phase of the cell cycle, so the donor cells are starved to put them in a nondividing stage making them compatible. 2. Enucleation of unfertilized eggs.-the chromosomes from an unfertilized egg (oocyte) are removed using a micropipet. 3. Nuclear transfer by cell fusion.-after enucleation, a micropipet is used to inject the donor cell into the cavity between the egg's plasma membrane and the noncellular membrane surrounding the egg. The two cells are usually fused by elctrofusion. 4. Implantation of the embryo into a surrogate mother and analysis of clones

• Be able to list the four important features common to all cloning vectors

1.Replicate independently. 2.Contain a number of restriction endonuclease cleavage sites that are present only once. 3.Carry a selectable marker. 4.Relatively easy to recover from host cell.

oligonucleotide probe

A labeled short sequence of nucleotides chemically synthesized to have the same or similar sequence to that of a specific gene or DNA sequence of interest, such that the denatured probe and target DNA can hybridize when they are renatured together

degenerate probe

A mixture of all the oligonucleotides that can encode a selected portion of a peptide sequence.

temporal expression

A pattern of gene expression in which a gene is only expressed during a specific time in development.

end-labeling

A rapid and sensitive method for radioactively, or nonisotopically, labeling DNA fragments and is useful for visualizing small amounts of DNA; involves either adding a labeled nucleotide to the 3' hydroxyl end of a DNA strand or exchanging the unlabeled nucleotide to the 3'-hydroxyl end of a DNA strand or exchanging the unlabeled 5' phosphate group for a labeled phosphate.

• Know what fusion proteins/affinity tags are, be able to give examples and explain what they're used for.

Affinity tag- an amino acid sequnecne that has been engineered into a protein to allow for its purification by affinity chromatography. The tag could be an entire protein or a short peptide sequence. These are often attached to reporter gene to ensure that only the reporter protein is made or that the reporter protein is fused to another protein. Commonly used protein tags are 6-histidine, glutathione-S-transferase, transcription factor c-Myc, FLAG, and influenza A virus hemagglutinin (HA). 233

• Be able to match model organisms to the area of research they are known for.

Bacteriophage lambda (l) The bacterium Escherichia coli Slime mold: Dictyostelium discoideum • cell-cell communication Ciliate: Tetrahymena thermophila • Telomere biology Yeast: Saccharomyces cerevisiae and Schizosaccharomyces pombe • cell cycle studies due to 90 minute cell cycle Worm: Caenorhabditis elegans • Genetic regulation of organ development, apoptosis Fly: Drosophila melanogaster • Genetics, body structure development Fish: Danio rerio • Development and pathology of animals Plant: Arabidopsis thaliana • Model plant Mouse: Mus musculus • Study of human disease, similar DNA Frog: Xenopus laevis and Xenopus tropicalis • Study of induction

• Be able to list and explain the steps involved in PCR

Basic requirements for in vitro DNA synthesis: •DNA polymerase •DNA template •Free 3′-OH to get the polymerase started •dNTPs Three steps of the reaction performed in an automated thermal cycler •Denaturation of the template DNA (e.g. 95C). •Annealing of primers (e.g. 55-65C). •Primer extension by a thermostable DNA polymerase (e.g. 72C). Denaturing - when the double-stranded template DNA is heated to separate it into two single strands. Annealing - when the temperature is lowered to enable the DNA primers to attach to the template DNA. Extending - when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

• Be able to explain the use of selectable marker genes in molecular cloning.

Basically a vector may have a gene that is responsible for resistance to ampicillin. the non transformed cells dont have it so they aren't resistant and their growth will be inhibited on agar with ampicillin. A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes; bacteria that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those bacterial colonies that can grow have successfully taken up and expressed the introduced genetic material.

• What limitations/pitfalls are there when cloning mammals?

Cloning animals for medical uses are weighed against ethical and financial costs. Currently the technology has low efficiency, less than 1-10% of cloned mammalian embryos results in offspring. There are many birth defects observed in cloned mammals after birth. When 10,000 genes were screened, 4% were shown to not be functioning correctly. Cloned animals suffer from developmental abnormalities such as extended gestation, large birth weight, inadequate formation of the placenta, historical defects in organs such as the kidney, brain, cardiovascular system, and muscle. There is a tendency toward obesity, liver failure, pneumonia, and premature death.

RNA-interference

Control of gene expression by specific mRNA degradation or translational repression caused by insertion of a double-stranded RNA into a cell

• Be able to list and be familiar with the methods used for monitoring DNA-Protein interactions.

EMSA- principle that protein-DNA complexes move more slowly than unbound DNA. Often called gel-shift mobility test DNase I footprinting- used to precisely map DNA-protein binding sites CHIP- important technique used for studying DNA-protein or RNA-protein interactions in vivo page 228

western blotting

Electrophoresing proteins, then blotting them to a membrane and reacting them with a specific antibody or antiserum. The antibody is detected with a labeled secondary antibody. This technique is used to identify and locate proteins based on their ability to bind to specific antibodies.

• Be able to list/ explain the steps involved in cloning and expressing a gene in bacterial cells.

Five major steps for molecular cloning using a plasmid vector: 1.Construction of a recombinant DNA molecule. 2.Transfer of ligation reaction products to host bacteria. 3.Multiplication of plasmid DNA molecules. 4.Division of host cells and selection of recombinant clones, e.g. by blue-white screening. 5.Amplification and purification of recombinant plasmid DNA. Transformation: transfer of recombinant plasmid DNA to a bacterial host Bacterial cells are incubated in a concentrated calcium salt solution to make their membranes leaky. The permeable "competent" cells are mixed with DNA to allow DNA entry. Alternatively, a process called electroporation drives DNA into cells by a strong electric current. From book: First, the DNA fragments to be cloned are generated by using restriction endonucleases. Second, the fragments produced by digestion with restriction enzymes are ligated to other DNA molecules that serve as vectors. (vectors replicate independently) Third, the recombinant DNA molecule is transferred to a host cell. Within this cell the recombinant DNA molecule replicates, producing dozens of identical copies known as clones. As the host cells replicate, the recombinant DNA is passed on to all progeny. Finally, the cloned DNA segments can be recovered from the host cell, purified, and analyzed. From the gene cloning in plain English video: 1. Isolate the target gene. 2. Insert target gene into a plasmid. 3. Insert the plasmid into bacteria. 4. Grow the bacteria.

FRET

Fluorescence Resonance Energy Transfer- a method for detecting protein-protein interaction in situ by nonradiative process whereby energy from an excited donor fluorophore is transferred to an acceptor fluorophore that is within approximately 10 no of the excited fluorophore.

• Understand the difference between homologous and nonhomologous recombination of transgenes in the making of knockout mice. Know what the neo and TK genes are used for and how they indicate which type of recombination has taken place.

Homologous recombination- by a mechanism that is poorly understood, the targeting vector finds the targeted gene and recombination takes place within the flanking homologous sequences. The end result is that the neo gene (positive selection marker) is inserted into the chromosome in the place of a target mouse locus. The result is cells that grow in neomycin and ganciclovir Nonhomologous recombination- if the targeting vector aligns in a nonhomologous region of the mouse genome, then recombination is rando nans both the positive (neo) and tk selection markers are integrated into the genome. The products of non-homologous recombinant are cells that are resistant to neomycin but are killed by ganciclovir. Neo gene- encodes for the enzyme neomycin phosphotransferase and serves as a marker to indicate that the vector DNA was inserted into the mouse chromosome Tk- a second marker that is attached to the targeted marker; it is inserted outside of the area of the endogenous gene and breaks down ganciclovir and produces a byproduct that it toxic to the animal

ligation reaction

In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors - carrier DNA molecules that will replicate target fragments in host organisms.

• Be able to explain why inducible expression systems are used in making transgenic animals

In some cases, introduction of a transgene results in death or such reduced viability. Inducible expression systems allow for the researcher to maintain the transgene line. An example of this is the Tet-off and is the a commonly used inducible expression system in which the transgene expression is dependent on the activity of an inducible transcription activator. Expression of the gene can then be regulated both reversibly and quantitatively be exposing the animals to various concentrations of antibiotics such as tetracycline and its derivative.

• Know the different types of chromatography and how they can be used to purify molecules.

Liquid Chromatography -Molecules dissolved in a solution will interact (bind and dissociate) with a solid surface. When the solution is allowed to flow across the surface, molecules that interact weakly with the solid surface will spend less time bound to the surface and will move more rapidly. Commonly used to separate mixtures of nucleic acids and proteins. 3 main techniques Gel filtration chromatography: separation by differences in mass. •Ion-exchange chromatography: separation by differences in charge. •Affinity chromatography: separation by differences in binding affinity.

EST-based probe

Made of partial cDNA sequences generated by amplifying cellular mRNA by PCR; these may be used to identify gene transcripts and are instrumental in gene determination and gene sequence discovery

• Be able to list the different methods used in labeling nucleic acids

Method depends on application: -Internal (uniform) labeling or end labeling? -Radioactive or nonradioactive? •Labeling involves DNA or RNA synthesis reactions or other enzyme-mediated reactions. Some methods for labeling nucleic acids: •Random primed labeling •In vitro transcription •Klenow fill-in oligonucleotide labeling

• Be able to explain the steps involved in making a transgenic mouse using pronuclear microinjection.

Microinjection of DNA into the pronucleus of a fertilized mouse egg. Implantation of the microinjected embryo into a foster mother. Analysis of mouse pups and subsequent generations for the stable integration and expression of the transgene.

• Be able to list and be familiar with the methods used for monitoring transcription and/or RNA levels.

Northern blotting, in situ hybridization, ribonuclease protection assay, RT-PCR (these are all explained in terms) for monitoring mRNA-Northern blot In situ hybridization RNase protection assay (RPA) Reverse transcription-PCR Quantitative real-time PCR (Q-PCR)

first-strand synthesis

Process in which reverse transcriptase adds dNTPs from 5' to 3' by complementary base pairing

• Be able to list and be familiar with the methods used to detect Protein-Protein interactions.

Pull down assay- used for analysis of protein-protein interactions in vitro. For example, a GST pull down assay tests interactions between a GST-tagged protein called the bait and a monitor protein called the prey. Yeast two hybrid assay- used to monitor in situ. One protein (bait) is produced from a fusion protein with a DNA-binding domain from another protein. The other protein (prey) is produced from a fusion protein with a transactivaton protein. If the two interact with each other, they produce a transcriptional activator that can activate one or more reporter gene Coimmunoprecipitation assay- monitors in vivo. Proteins from cell extracts are reacted with a specific antibody or antiserum against one of the proteins, then immunoprecipitated by centrifugation. The precipitated proteins are usually detected with a Western Blot. FRET- see terms

• Be able to explain blue-white screening and red-white screening.

Red-white screening- Host yeast strain: ura3/trp1/Ade2-1 mutant •When foreign DNA is inserted in the multiple cloning site, SUP4 expression is interrupted. -The Ade2-1 mutation is no longer suppressed. •ADE1 and ADE2 encode enzymes involved in adenine biosynthesis. •Ade2-1 mutant cells produce a red pigment from polymerization of an intermediate compound. •In the absence of foreign DNA, SUP4 is expressed. -The Ade2-1 mutation is suppressed. •Ade2-1 mutant cells expressing SUP4 are white (the color of wild-type yeast cells). In the case of the vector pUC18, blue-white screening is used to distinguish recombinant from nonrecombinant transformants. •Also known as "lac selection" or - complementation More for blue white screening- Figure 8.7.

• Understand how to use restriction mapping to characterize DNA

Restriction mapping provides a compilation of the: •Number of restriction endonuclease cutting sites along a cloned DNA fragment. •Order of restriction endonuclease cutting sites. •Distance between restriction endonuclease cutting sites.

• Be able to explain how restriction endonuclease enzymes are used by bacteria to keep from being transduced by bacteriophages (the modification and restriction systems).

Restriction system- "Restrict" or prevent viral infection by degrading the invading nucleic acid. Modification system-Methylase activity: Addition of methyl groups to protect those sites in DNA sensitive to attack by a restriction endonuclease. (Helps host from being chewed up) •Typically adenine methylation (6-methyl adenine). •Methylation pattern is maintained during DNA replication.

• Know what techniques are used to detect transgene incorporation into mouse genomic DNA.

Tail biopsies are taken from mouse pups to obtain DNA for analysis. The DNA is tested for the presence of the transgene by Southern blot hybridization or PCR. A mouse in which there is successful integration is referred to as a founder of a new transgenic lineage.

• When cloning a gene into a vector, how can you keep the vector from annealing to itself?

The degree of self ligation can be reduced by treatment of the vector with the enzyme phosphatase, which removes the terminal 5' phosphate resulting in the inability to recircularize because there is nothing to make a phosphodiester bond. 5' phosphate is provided by foreign DNA. Another strategy is to use two different restriction endonuclease cutting sites with non complementary sticky ends. This inhibits self-ligation and promotes annealing of the foreign DNA in the desired orientation within the vector.

• Be able to list the minimum requirements for transgenic constructs

The transgenic construct at minimum contains a promotor element, complementary DNA (cDNA ) for the gene of interest, and a polyadenylation signal.

• Be able to explain how ddNTPs are used in DNA sequencing

They are used in the "sanger" or "dideoxy" method of sequencing, which is in essence a DNA synthesis reaction. in this method single stranded DNA is mixed with radioactively labeled primer to provide the 3' OH required for DNA polymerase to initiate DNA synthesis. ddNTPs are replication terminators because they are lacking the 3'OH and cant form a phosphodiester bond with another nucleotide. Thus the reaction proceeds until replication-terminating nucleotide is added.

• Understand why the CRE/LOX system is used.

This system is used because some gene knockouts result in death. Conditional mutations are used to studuy a gene's role at any time of development. The Cre/Lox system is an example and is based on site-specific DNA recombination. This system makes it possible to manipulate genes in eukaryotes by means of activation of transgene expression by site-specific recombination.

• Know the different classes of restriction enzymes

Types I and III not useful due to random cutting patterns •Type II restriction endonucleases are widely used by molecular biologists. -Most can separate endonuclease and methylation functions Table 8.1

• Be able to list and be familiar with the methods used to monitor translation and/or protein levels.

Western blot.- see terms In situ analyses.- Page 244 e.g. indirect immunofluorescence assay- fluorescently labeled secondary antibodies to the primary antibody against the target protein Enzyme-linked immunosorbent assay (ELISA)- an example of this is the sandwich ELISA. The Sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) is a sensitive and robust method which measures the antigen concentration in an unknown sample. The antigen of interest is quantified between two layers of antibodies: the capture and the detection antibody. These antibodies must bind to non-overlapping epitopes on the antigen. Southern blot- see terms Constructing fusion proteins with an easy-to-detect tag.

RT-PCR

a PCR method that begins with the synthesis of cDNA from an mRNA template, using reverse transcriptase. The cDNA then serves as the template for conventional PCR

reporter gene

a known gene whose RNA or protein levels can be measured easily and accurately

RNA probes

a labeled RNA generated by in vitro transcription from a cloned DNA sequence that is complementary to a specific gene or DNA sequence of interest, such that the riboprobe and target DNA can hybridize when they are renatured together.

x-ray crystallography

a method for determining the three-dimensional structure of molecules by measuring the diffraction of X-rays by crystals of a molecule or molecules.

constitutive expression

a pattern of gene expression in which a gene is expressed at all times.

spatial expression

a pattern of gene expression in which a gene is only expressed in specific tissues of an organism.

micro ballistic transfection

a physical method of cell transformation in which high-density, sub cellular-sized particles are accelerated to high velocity to carry DNA or RNA into living cells via a "gene gun"

homologous probes

a probe that is exactly complementary to the nucleic acid sequence of interest

heterologous probes

a probe that is similar to, but not exactly the same as, the nucleic acid sequence of interest

totipotent

cell that has the capacity (total potential) to form an entire organism

pluripotent

cells capable of giving rise to all cell types because they are not yet specialized

genomic library

contains DNA fragments that represent the entire genome of an organism

FISH

fluorescent in situ hybridization a process which hybridizes a fluorescent probe to whole chromosome to determine the location of a gene or other DNA sequence within a chromosome. THis is useful for identifying chromosomal abnormalities and gene mapping. Also used to determine the precise localization of RNA transcripts within a cell

internal labeling

form of labeling nucleic acid; provides maximal labeling and is often used to screen libraries or for Southern blots, this method is preferred over end-labeling

tissue-specific genes

genes activated only in particular cell types a set of genes whose expression or function is necessary for cell differentiation into a cell or tissue type; muscle, nervous system

housekeeping genes

genes alive in most cell types because they are required for basic functions such as protein synthesis or maintenance of the cytoskeleton

transfection

introduction of foreign DNA into eukaryotic cells, either for a short duration (transient) or for long-term analysis (stable integration)

• Be familiar with the different types of reporter genes and understand what they are used for.

lacZ (lac operon)- bacteria. product is B-Gal. its a widely used reporter system. the enzyme hydrolyzes the colorless substrate X-gal to a blue precipitate for localization of gene expression in situ. luc-firefly. product is luciferase. highly sensitive reporter system that oxidizes luciferin and generates a bioluminescent product (photons) cat- a useful reporter for in vitro assays, but protein gives poor resolution in situ gus-generally used reporter in plant systems gfp- a reporter that fluoresces on irradiation with UV; because it is autofluorescent, it has the distinct advantage that it can be used for imaging live cells.

morpholinos

modified DNA analogs with an altered backbone linkage that lacks a negative charge; used in antisense-mediated inhibition of gene expression

antisense oligonucleotides

short oligonucleotides designed to selectively bind to a specific mRNA and induce antisense-mediated inhibition of gene expression. Either the mRNA strand in the hybrid duplex is cleaved by RNase H or translation is blocked.

• Understand how GC content affects the hybridization of probes

stability of the hybrid duplex or "heteroduplex" is influenced by the number of hydrogen bonds between the bases and base stacking by hydrophobic interactions that hold the two strands together.

in vitro

studies performed in cells or tissues grown in culture, or in cell extracts or synthetic mixtures of cell components

in vivo

studies performed within a living organism

transformation

the introduction of a foreign DNA into bacterial cells

knockin mouse

the process of gene targeting by homologous recombination is used to introduce mutations in the coding region of a gene

knockdown mouse

the process of gene targeting by homologous recombination is used to introduce mutations in the regulatory elements of a gene to inactivate or modify expression levels of that gene

transduction

the transfer of DNA from one bacterium to another by a bacteriophage

electroporation

the use of a strong electric current to introduce DNA into cells

northern blotting

transfer of RNA fragments to a support medium. A technique used to identify and locate mRNA sequences that are complementary to a piece of DNA (or antisense RNA) called a probe.

gene pharming

turning animals into pharmaceutical bioreactors for protein-based human therapeutics/the use of transgenic animals to produce recombinant pharmaceutical proteins in their milk of eggs

stem cells

undifferentiated cells that can undergo unlimited division and can give rise to one or several different cell types

subcloning

when previously isolated clones are transferred into a different vector for other applications

• Know the difference between stable and transient transfection.

• Transient transfection: the introduction of DNA into cells for a short duration. • Stable transfection: Cells that have stably integrated the plasmid into a chromosome are selected for by drug resistance.