system the code for life organism organ tissues cell. nucleus

System

The Code For Life

Organism

Organ

Tissues

Cell

.

Nucleus

Nucleus

The Code For Life

Chromosome

Genes

Brown eyes

Straight hair

Big nose

A cell is an organization of millions of moleculesA cell is an organization of millions of molecules

Proper communication between these molecules Proper communication between these molecules is essential to the normal functioning of the cell is essential to the normal functioning of the cell

To understand communication between To understand communication between

molecules:molecules: *determine the arrangement of the atoms*

Organ Organ Tissue Tissue Cell Cell Molecule Molecule Atoms Atoms

Structural BiologyStructural BiologyMedicine and Biology at the Atomic ScaleMedicine and Biology at the Atomic Scale

Advanced Cell & Developmental Biology

Gene, Recombinant DNA & Cloning Analysis

Restriction Enzymes

• Restriction enzymes are DNases (nucleases) found in bacteria that recognize specific DNA sequences as 4mers,6mers or 8mers and make double stranded breaks in DNA .

• This enables cutting of genome in specific ways to generate restriction site maps and the development of approaches for pasting pieces of DNA together in specific ways.

A

B

C

D ,E

F

Separation of EcoR1 segments on an agarose gel

DNA Hybridization

• DNA hybridization is the process whereby complementary strand of DNA anneals (to form a double helix) with the single stranded DNA

• Hybridization can be measured by labeling the “complementary strand” either with 32P nucleotides or fluorescent probes .

• There is also DNA-RNA hybridization

Southern Blotting

• Southern Blotting enables identification of specific DNA sequences (gene

fragments) from among the total sequence of DNA

Cut DNA with restriction enzymes

Separate fragments on agarose or acrylamide gels

Transfer the separated DNA from gel on to nitrocellulose paper

Hybridize with a labeled DNA or RNA of interest ( e.g., 32P labeled DNA) followed by autoradiography or phosphoimaging for detection

Northern Blotting• Northern Blotting is where RNA is blotted and then probed labeled DNA (cDNA) synthesized from the mRNA isolated from the cell

• Enables identification and quantification of specific mRNAs from among the vast

population of RNAs in the cell

DNA cloning

• DNA cloning enables specific pieces of genome to be inserted into bacteria as plasmid or phage lambda vectors and grown in large quantity.

• The first step is to generate a library of bacteria with inserted DNA fragments. This could either be a genomic(DNA)or a cDNA (mRNA) library

Replica plating and in situ hybridization

• Techniques used to identify a bacterial colony that contains the gene (DNA sequence)

of interest. The isolated colony can be grown up in large quantities.

Replica plating and in situ hybridization

CsCl centrifugation for separation of plamid DNA from chromosomal DNA

cDNA libraries

• They are generated to isolate particular genes of interest or to identify a gene based on the protein expression of that gene cloned in the bacterial cell

• The latter procedure is called “reverse genetics” whereby the protein product is used to identify the gene followed by DNA sequencing

DNA sequencing

• Sanger’s dideoxy method DNA to be sequenced is mixed with each of 4 ddNTPS (chain terminators) in separate reactions for DNA synthesis and later separation of the products by electrophoresis

• Can now be done automatically via sequencing machines that work with different flurochromes attached to each of dideoxy nucleotides

• To determine the sequence of a gene of many kilobases overlapping DNA fragments of 400-800 bp must be sequenced

Protein expression vectors

• These are specially designed plasmid

vectors for fusion protein expression

to isolate large quantities of protein of

interest for antibody production or

other studies of purified protein.

• The proteins are produced as fusion

proteins of the cDNA gene coding

sequence ligated to a protein

expression marker or reporter protein

e.g. beta-galactosidase

• They can also be used as a major tool

in cell biology to study the expression

of proteins in cells following DNA

transfection

DNA transfection and Polymerase chain reaction (PCR)

• DNA transfection is used to track the properties of individual proteins in a cell

Construct a plasmid expression system that contains the protein of interest fused with a reporter gene such as a beta- galactosidase or a short peptide sequence such as HA 9 mer peptide or FLAG epitope for antibody localization with anti HA or anti FLAG or fluorescent localization in living cells with GFP-constructs (GFP-actin)

Polymerase chain reaction (PCR)Is used as an alternative to cloningfor purifying a particular DNA (genesequence

It enables the production of microgramquantities of the DNA sequence of interest in the test tube

Provides an alternative for preparing DNA probes to screen genomic or cDNA libraryfor clones encoding a protein of interest

DNA Microarrays and chips

• Enable via fluorescence in situ hybridization (FISH) to measure expression of 1000’s of genes on each array/ chip.

Yeast genome microarray: The array is hybridized to cDNA labeled with a green fluorescent dye prepared from cells grown in glucose and with red labeled cDNA from cells grown in ethanol. Spots were detected with a scanning confocal microscope

Actual chip size

Antibody production

• Polyclonal antibodies are

generated by injecting

antigen into an animal and

purifying the antibody

titer from blood

• Monoclonal antibody

technique enables to obtain

a single clone of cells that

recognizes one epitope

( usually ~ 9 a.a.) of the

total protein

Monoclonal antibody production

Genetic Engineering

• Introduction of exogenous genes ( mutant or normal) in to normal cells or organisms to study gene expression

• Used to study the role of the protein coded by the gene in the cell/organism function or for engineering gene expression for improving food production or reducing the destrcutive damage of human diseases

Site Directed Mutagenesis

• Alterations in nucleotides (substitutions or deletions) in vitro at known (directed) sites to create “mutant genes”

• These mutant genes can be transfected into cells as previously discussed and enables study of gene function at the individual cell level. The transfected genes are also called “transgenes”

Production of transgenic mouse

Inject mutant gene in to one of the pronuclei of the fertilized mouse oocyte

Transfer oocyte to surrogate mother. 10-30% of offspring contain the transgene in equal amounts in all tissues

Gene Knockout or “replacement”

• Form of trangenics

• Occurs following homologous recombination of the transgene at the site of the endogenous gene

• Occurs readily in yeast cells but in mammalian cells the rate of recombination is very slow and hence a double selection marker approach is adopted where the first marker e.g. neomycin resistance selects for all cells with homologous recombination while the second marker allows growth of only those cells that carried out homologous recombination

Knockout protocol

ES cells are isolated from the inner blastocyst and culture

ES cells are tranfected with the gene of interest

ES cells successfully transfected via homologous recombination are selected and grown in culture and injected into a host blastocyst. Chimeras develop which contain ES cells from both the transfected and the host cells.

Enables direct study of gene function in an intact organism

Gene Replacement/therapy

• Replace an abnormal gene with a normal one at a very early stage of development

• It has the potential for curing or alleviating the symptoms of a wide variety of human diseases, e.g.,Parkinson’s disease

Procedure for gene replacement

How Ian Wilmut Made Dolly 1Making Quiescent Cells

Finn Dorset ewe3.5 months pregnant

Mammary gland cells

Culture mammary cells

Harvest quiescent cells

Starve cells

Suction

Suction Pipette

Glass pipette

How Ian Wilmut Made Dolly 2Collecting The Donor Nucleus

How Ian Wilmut Made Dolly 3Egg Preparation

Scottish Blackfaced ewe egg donor

An egg is collected then placed into a dish where it can be manipulated

Egg

Suction

Suction Pipette

Glass pipette

EggChromosomes


Egg

Suction

Suction Pipette

Glass pipetteChromosomes


Suction

Suction Pipette

Glass pipette

How Ian Wilmut Made Dolly 4Inserting The Donor Nucleus

Suction

Suction Pipette

How Ian Wilmut Made Dolly 4Inserting The Donor Nucleus

How Ian Wilmut Made Dolly 5Initiating Development

Zygote


Cleavage


Morula


Scottish Blackfaced ewe surrogate

mother

How Ian Wilmut Made Dolly 6Development

Morula

Finn Dorset lambDolly

system the code for life organism organ tissues cell. nucleus

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