II. Methods of Studying Bioregulation The Scientific Methods: Controls; Reprensentative sampling; Dose-response relationship; Biological rhythms Methods

II. Methods of Studying Bioregulation

• The Scientific Methods: Controls; Reprensentative sampling; Dose-response

relationship; Biological rhythms

• Methods of Analyzing Endocrines: Extirpation-observation & Replacement-observation; Imaging;

Quantitation of hormones; HPLC; Immunohistochemistry; Analysis of hormone receptors

• Molecular Methods: Genetic and genomic approaches; transgenic animal

approach; knock out and knock down approaches Transcriptomics and Protiomics

• Statistics

Scientific Method

• Data; Hypothesis; Theory; Law or Principle

• Control experiments

• Representative sampling

• Dose-response relationship

• Biological Rhythms

Controlled Experimental Testing

• Variable: An event or condition that is subject to change

• Independent variable: the change or manipulation to the experiment

• Dependent variable: variable resulted from a change in the independent variable

• Experimental group vs. control group

• Control group: Untreated control Vehicle treated or sham operated control Initial control Placebo; double-blind study; control site; reference site

Importance of Initial Controls

• Comparison of data for animals taken at the beginning of the experiment (initial controls) allows one to determine whether the experimental treatment stimulated the animals (A) or simply maintained the initial conditions (B) while the “controls” declined.

Comparison of Adequate and Inadequate Controls

Treatment ITreatment II

Treatment III Treatment IVAdequate

controlInadequate

control

One independent variable: Surgery

Surgically altered animal

Sham-operated animal

Unaltered animal

One independent variable: Injection

Animals injected with bioregulator in vehicle

Animal injected with vehicle only

Uninjected animal

Two independent variables: Surgery and injection

Surgery and bioregulator in vehicle

Sham-operated plus biotergulator in vehicle

Surgery plus vehicle only

Sham-operated puls vehicle only

Dose-Response Relationship

A. A system may respond to a threshold dose and then continue to show increased response with increasing dose until the process reaches a maximum

B. Relationship of optimal response to an “optimal” dose. Any dose above or below that will produced reduced effect

C. Species-specific or tissue-specific dose–responses. D. J-shaped or U-shaped dose–response curves are common in biological

systems when dealing with very low doses.

• Sub-threshold dose• Threshold dose• Observed effect dose• Paradoxical nature• Dose-response curve

Dose-Response of TCDD on Levels of GH and PRLmRNA Expression

This is a study conducted by Elango et al. (2006) to determine the effect of TCDD (an environmental pollutant) on the levels of PRL and GH mRNAs in the pituitary glands of rainbow trout cultured in vitro. An unique dose-response curve was observed

Reference: Elango et al. (2006), General and Comparative Endocrinology 145, 116-127, 2006.

Diurnal Pattern of Plasma Corticotropin and Cortisol

• The release of hormones or other bioregulators offen occurs in burst (phasic secretion) rather than at a continuous and constant rate (tonic secretion)

• Many of these bursts exhibit distinct, predictable diurnal, monthly, or seasonally cyclic patterns of secretion (biological rhythms)

• The example below shows the diurnal patterns of secretion of ACTH and cortisol in human

Methods of for Studying Endocrines

• Extirpation-Observation and replacement-observation

• Imaging

• Radioimmunoassay (RIA) and quantitative enzyme –linked immunoabsorbant assay (ELISA)

• High-performance liquid chromatography analysis

• Immunocytochemical analysis

• Techniques for determining the nature and characteristics of receptors

Extirpation-Observation & Replacement-Observation

• Early studies of hormone actions were limited to gross manipulations and observations. Putative regulators were identified by first removing a gland or other organ and observing the effect of removal of the gland or organ. By replacing the lost tissue or gland through transplantation or grafting or administering the extract prepared from the tissue (or gland) and observe whether the effects are restored

• Examples:– In insects, there are two hormones regulating insect

development. These two hormones are juvenile hormone and ecdysone. The functions of these two hormones were found by “Extirpation-Observation and Replacement-Observation experiments to be discussed in the next few slides

Functions of Juvenile Hormone and

Ecdysone

Application of Ligature to Determine the Presence of Ecdysone in Insects

Ecdysone is produced in the prothoracic gland located in thoracic area

Ligature was applied to larvae of lipeloptora in the 3rd instar inter-molt stage to confine prothoracic gland in one portion of the animal

When the animal goes through the process of metamorphosis (molting), the portion of the animal that contains the prothoracic gland will molt into pupa and the other part remains as larva

To confirm these results, prothorasic gland can be injected into the ligated larva and observe the outcome of the animals

B

Effect of Implanting Corpus Allatum (CA) on Metamorphorsis of Rhodnius

A: 5th instar Rhodnius B: Adult Rhodnius C: 6th instar produced by implanting CA from 4th instar

into the abdomen of the 5th instar D: 6th instar animal like C but with intermediate wing

structure

Parabiosis Decapitated 5th instar Rhodnius was

jointed to 4th instar Rhodnius By pumping both body parts to allow

mixing of the hymorlmph between both animals

After molting, the body of the 5th instar Rhodnius molted into 6th instar (still remains the characteristics of instar but not adult)

This experiment suggested that the juvenile hormone produced by the CA of the 4th instar Rhodnius controlled the 5th instar body part to molt into 6th instar rather than molted into the adult

In fish, the technique of hypophysectomy (removal of pituitary gland) has been used to demonstrate the presence of pituitary hormones

Levels of Hormones in Human Serum

pg/ml

Physiological dose (ng/ml, pg/ml, fg/ml) vs. pharmacological dose (g/ml or higher)

Methods for Determining Levels of Hormone

• Bioassay system: Animals or parts of the animal that can provide a qualitative or quantitative estimation of the hormonal factors

• Radioimmunoassay (RIA): Developed by Rosalind Yalow and Solomon Berson while study insulin. This technique was awarded with a Nobel Price for recognizing the discovery by Yalow and Berson This technique relies on the availability of pure hormone, radio-

labeled hormone and a specific antibody

• Quantitative enzyme–linked immunoabsorbant assay (ELISA): This technique is similar to RIA except the hormone does not have to be radio-labeled, and the antigen or antibody is fixed on a solid surface

• Both techniques can detect hormone at the concentration at pg level or lower.

Rat Tibia Bone Used in Sulfation Assay

• The growth point of the rat tibia bone was used as an assay system for estimating the biological activity of IGF-I and growth hormone

• During the growth of the tibia bone, incorporation of radioactive sulfate into glycoproteins in bone which can be detected. This is the basis of sulfation assay

• By measuring the longitunal growth of the bone directly, the activity of GH oe IGF-I can also be determined

• PZ: Proliferative Zone

• HZ: Hyperative Zone

• PS: Primary spongiosa

• Tibia bone growth assay

• Sulfation assay

Reading Assignment1. Lama et al., 2000. Recombinant human GH enhances tibial

growth in peripubertal females but not males. Europ. J. Endocrinol. 142: 517-523.

Fish Gill Cartilage Assay to Determine Growth Hormone Level (I)

Reading Assingment: Cheng and Chen, 1995. Synergism of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in stimulation of sulphate uptake by teleostean bronchial cartilage in vitro. J. Endocrinol. 147: 67-73.

The soft bone in the fish gill arch cartilage can uptake 35S-sulfate into the cartilage in response to IGF-I administration. This is because IGF-I can stimulate mitogenesis in the growing point of gill arch. Cheng and Chen (1995) found that GH can stimulate IGF-I to a highher extent of mitogenic activity

Fish Gill Cartilage Assay to Determine

Growth Hormone Level

A linear relationship can be identified

A Novel Bioassay for Serum GH

• A Ba/F3-hGHR cell line was developed from a mouse pro-B cell lymphoma by transferring GH receptor gene. This cell line shows a dose-dependent proliferation induced by GH as shown in the above figure

• The dose dependent range is: 1 ng to 100 ng/ml

• Reading assignment: Ishikawa et al., 2000. A novel specific bioassay for human GH. J. Clinical Endocrinol. 85: 4274-4279.

• Radioimmunoassay (RIA): Developed by Rosalind Yalow and Solomon Berson while study insulin. This technique was awarded with a Nobel Price for recognizing this important discovery by Yalow and Berson in 1977

Radioimmunoassay to Determine Levels of Hormone

Principle of RIAUnlabelled antigen competes with radiolabelled antigen (usually labeled with 125I) for the limited binding sites available on the antibody molecule made specifically for the antigen of the interest. At the end of reaction labeled antigen bound to antibody is separated and measured for the radioactivity. Amount of the radioactivity separated is compared with values of known antigen standards and the concentration of antigen presents in the sample is calculated.This technique relies on:Purified & radiolabeled antigen (hormone)Specific antibody to the hormone

Labeling Protein Hormone by Iodination

• Iodination method is based on oxidizing radioactive iodide (I-) to I+ which then spontaneously incorporates into tyrosine residues. This method is particularly used to label proteins

• There are several oxidizing agents commonly used in iodination reaction: Chloramine T Iodogen: 1,3,4,6 Tetrachloro-3a,6a-diphenyl-glycduril Pierce iodination reagent

Peroxide and lactoperoxidase

Separation of 125I-Labeled Protein by Chromatography on Sephadex G-100 Column

• The unincorporated radioactive I needs to be separated from the labeled product by gel filtration or other methods

• Antigen could be altered by the process of labeling, and therefore need

to be confirmed by: Gel filtration separation Biological activity assay

Determine the Amount of Radio-labeled Antigen for the Assay

The optimal amount of radio-labeled antigen to be used in the assay is typically enough to bind approximately 50 % of the added antibody (which is the same in each tube).

RIA: Standard Procedure

• Set up multiple tubes for standard and unknown samples (sufficient tubes for triplicates)

• Add fix volume of buffer to each tube

• Add various known amounts of unlabeled bioregulator to each tube (These will compete the binding sites of the antibodies)

• Add fix amount of radio-labeled bioregulator to each tube

• Add fixed amount of antibody to each tube and incubate the mixture at cold for 24 h or longer until it reaches equilibrium

• Separate the bound radio-labeled antigen with the un-bound antigen by:Charcoal-dextrin absorption;Binding of the antibody to the tube;Precipitation with the second antibody; orPrecipitation with ammonium sulfate

• Determine the radioactivity of the complex

• Plot the results as:% bound radioactivity vs. bioregulator concentration; or ratio of bound/free vs. antigen concentration

Establishment of a Standard Curve

bioregulator

...

bioregulator

Reading Assignment:

1. Thomas et al., 1972. Radioimmunoassay of human growth hormone: technique and application to plasma, cerebrospinal fluid, and pituitary extracts

2. Baxter et al., 1985. the measurement of testostrone and oestradiol-17b using iodinated tracers and incorporating and affinity chromatography extraction procedure

3. Yalow 1977. Nobel Lecture on Radioimmunoassay

Principle of ELISA (Enzyme-linked Immunoabsorption Assay)

• Advantages of ELISA No radio-labeled

antigen is required ELISA can be used to

quantify antibody in a sample

ELISA can be used to quantify antigen in a sample

Large number of samples can be processed at a time.

Highly sensitive

• ELISA: Direct ELISA Indirect ELISA Competitive ELISA

bioregulator

Direct ELISA Indirect ELISA

Indirect ELISA

A Standard Curve

• Add bioregulator to be determined to each well and allow it to bind to the well

• Add antibodies to each well to bind to the bioregulator

• Wash each well with buffer, add enzyme linked second antibodies specific to the first antibody. Wash off the unbound second antibody

• Add substrate to each well to develop the color. Determine the intensity of the color

http://2.bp.blogspot.com/-n_d0Z2tej6M/T7J8y6aZPHI/AAAAAAAAAt4/OCypawg-fkc/s1600/elisa-1.jpg

1. Plate is coated with a capture antibody2. sample is added, and any bioregulator present binds to capture

antibody3. detecting antibody is added, and binds to bioregulator4. enzyme-linked secondary antibody is added, and binds to

detecting antibody5. substrate is added, and is converted by enzyme to detectable

form

Sandwich ELISA

http://en.wikipedia.org/wiki/File:ELISA-sandwich.svg

1. Bioregulator (Sample) is incubated with an unlabelled antibody, these bind each other to form bioregulator-antibody complex

2. These bound antibody/bioregulator complexes are then added to an bioegulator-coated well

3. The plate is washed, so that unbound antibody is removed,

4. A secondary antibody specific to the primary antibody is added. This secondary antibody is coupled to an enzyme

5. When a substrate is added, enzyme acts on the chromogenic substrate and emits colour

6. ELISA reader is used to measure the intensity.

Competitive ELISA

Fixed amount of antibody and various amount of antigen in each tube

Coat each well with fixed amount of bioregulator

Standard Curve

Identification of mRTP Cells Producing GH and PRL by Immunocytochemical Staining

1E

1F

Phc GHAb PrlAb

100 m

1. ELISA2. Sylvester-Hvid et al., 2002. Establishment of a quantitative

ELISA capable of determining peptide – MHC class I interaction. Tissue Antigen 59: 251-258.

Reading Assignment:

High Performance Liquid Chromatography (HPLC)

• This method is important for purification and identification of bioregulators

• Depenpending on different types of column used, HPLC can be used to identify and purify different bioregulators

• Sensitivity is at ng/L level

Reverse phase (hydrophobic) ; aqueous phase

Imaging

• Light microscopy, fluorescence light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal microscopy

• Tomography: whole-body scanning technique Computer axial tomography (CAT scan) ---CAT scan relies

on X-ray to construct two-dimensional slides that can be converted to a three-dimensional image by a computer

Magnetic resonance tomography (MRT scan) --- MRT scan relies on the bipolar nature of water molecules which can line up in a magnetic field. By this way, a computer can generate a three-dimensional image

Position emission tomagraphy (PET scan) --- PET scan employ the use of common metabolic molecules that are labeled with short-lived radioisotopes (e.g., 11C, 13N, 15O, 18F) to produce minimal radiation exposure to the subject.

Confocal Laser Microscopy

Application of confocal laser microscopy to the visualization of calcium movement in a single pituitary melanotrope cell. By vertically stacking successive images through the cell, a 3D image is created. Changes in fluorescence caused by calcium movement (red, high; blue, low) can be detected in multiple planes (X, Y, t) sliced through the cell.

Reconstruction from Multiphoton and Confocal

Microscopy

Elucidating the pattern and timing of pituitary LH (purple) and POMC (green) cells in the developing (A) and adult (B) mouse pituitary as reconstructed from multiphoton and confocal microscopy images. Panel C outlines the developmental sequence for cell appearance based upon the microscopic analysis. POMC cells first appear in the ventral anterior lobe (1) and then extend laterally and ventrally into the AL (2, 3). The first POMC cells in the intermediate lobe (melanotropes) appear next (4). LH cells then appear in the AL, first in the ventral AL (5) and then in the dorsal AL (6). Islands of melanotropes are found in the dorsal AL postnatally

Molecular Biological Tools Used in Studying Bioregulators

• Recombinant DNA technology has been used to identify genes or cDNA encoding bioregulators, or studying structures of peptide or protein bioregulators

• Reverse transcription (RT) – PCR or real-time RT-PCR allows the determination of levels of mRNA encoding for various protein bioregulators

• Transgenic animal technology allows studies for the determination of the in vivo biological functions of bioregulators

• Knock out or knock down animals allows studies to determine the in vivo biological function of bioregulators

• DNA microarray: Allows gene expreswsion profiling

• Proteomics including various techniques of protein separation, identification, quantification and structural analysis to study bioregulators

• Deep RNA sequencing and transcriptom

Reading Assignment:

1 Agellon and Chen, 1986. Rainbow trout growth hormone: molecular cloning of cDNA and expression in E. coli, DNA 5: 463-471

• Constructing cDNA library from RNA isolated from trout pituitary glands

• Isolating pituitary gland specific cDNA clones by competition hybridization

• Screening cDNA clones that produce GH by immunobinding assay

Total mRNA sscDNA dscDNA ft10 or gt11

cDNA libraryScreen the library byHybridization or immunoscreening

cDNA clones

RTDNA synthesis

Isolation of GH cDNA Clone by Radio-immuno-Binding Assay on Terazaki Plate

• Bacterial extract is coated on a Terazaki plate

• Block the plate with a buffer containing dry milk power

• Incubate the plate with GH-Ab

• Wash the plate with a buffer

• Incubate the plate with 125I-Protein A

• Wash the plate, autoradiography and develop the film

Reverse Transcription (RT) - PCR

• End Point RT-PCR: Measuring the presence of a particular mRNA Can be adapted to perform semi-quantitative

determination of an mRNA

• Real-time RT-PCR: For quantitative measurement of the level of an mRNA Absolute quantitative RT-PCR Comparative quantitative RT-PCR

Principle of RT-PCR

• Reverse Transcription:AAAAAATTTTTT

mRNA

Oligo dT

• PCR:

cDNA

Forward primer Reverse primer

PCR product

cDNA product

Reverse transcriptase

Taq DNA polymerase

PCR Amplification Program

• 1 cycle at 95 oC for 3 min to denature all templates

• 40-50 cycles: 95 oC for 15 sec for denaturation of PCR products At annealing temperature for 15 sec to anneal

primers to the templates 72 oC for 30 sec for synthesis of DNA

Regular PCR Machine

Measuring Levels of mRNA by Quantitative RT-PCR

• End point RT-PCR: Semi quantitative RT-PCR Competitive quantitative RT-PCR

• Real-time RT-PCR: Absolute quantitative real-time RT-PCR Relative quantitative real-time RT-PCR

For real-time RT-PCR analysis, the size of the PCR product should be within 100-200 bp, otherwise the data will not be precise

Determine Levels of mRNA by Semi-quantitative RT-PCR

• If levels of -actin mRNA in various tissues are constant, then the levels of mRNA of interest can be expressed in terms of relative to levels of -actin

• This assumption will only work when the increase of levels of mRNA is in the linear range of the detection

• Example given in the left of this slide is showing how the levels of NPY mRNA is determined by this method

Reading Assignment:

1.Greene and Chen, 1999. Quantitation of IGF-I, IGF-II, and multiple insulin receptor mRNAs during embryonic development in rainbow trout. Molecular Reproductive Development 54: 348-361.

Concept of Competitive Quantitative RT-PCR

Forward primer

Reverse primer

TargetCompetitor

PCR (fixed amount of target and various amounts of competitor

Analyze the product by electrophoresis on agarose gel

Various Amounts of Competitor

Example of Competitive Quantitative RT-PCR

In vitro transcription

Competitor mRNA

AAAA

IGF-II mRNA to be determined

ReverseTranscription

ReverseTranscription

Competitor cDNA IGF-II cDNA

Various amounts of competitor, target and fixed amount of amplification primers and proceed PCR amplification

Competitive Quantitative RT-PCR

When the ratio of target/competitor equals to 1, the levels of competitor equals the level of the target

• Green and Chen, 1999: Quantitation of IGF-I, IGF-II, and Multiple Insulin Receptor Family Member Messenger RNAs During Embryonic Development in Rainbow Trout, MRD 54: 384-361

Real-time PCR Machine

Principle of Real-Time RT-PCR

• SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light (λmax = 497 nm) and emits green light (λmax = 520 nm). The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower performance. SYBR green can also stain RNA with a lower performance than DNA.

• Since double stranded DNA can interact quantitatively with SYBR Green 1 preferentially and thus can be used to monitor the amount of DNA synthesized after each cycle of PCR reaction.

• Cycle threshold (CT): CT is defined as the number of PCR cycles required for the fluoescent signal to cross the threshold (i.e., exceeds background level). This is usually determined automatically by the real-time PCR machine set in the factory

Determination of Cycle Threshold (CT)

1 2 3 4 5 6 7 8 9 10 11 12

A

Content

Sample

Peak 1

Peak 2

B

Content Unkn-1 Unkn-1 Unkn-1 Unkn-5 Unkn-5 Unkn-5 Unkn-9 Unkn-9 Unkn-9

Sample

Peak 1 82.20 82.00 82.00 82.20 82.00 82.00 82.00 82.00 82.00

Peak 2 None None None None None None None None None

C


Sample

Peak 1 82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00


D


Sample

Peak 1 82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00


E


Sample

Peak 1 82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00


F

Content NTC-1 NTC-1 NTC-1

Sample

Peak 1 None None None

Peak 2 None None None

G

Content

Sample

Peak 1

Peak 2

H

Content

Sample

Peak 1

Peak 2

Sample Set Up in a 96-Well Plate

PCR Amplification Program

• 1 cycle at 95 oC for 3 min to denature all templates

• 40-50 cycles: 95 oC for 15 sec for denaturation of PCR products At annealing temperature for 15 sec to anneal

primers to the templates 72 oC for 30 sec for synthesis of DNA

• From the melting profile, one can determine whether the PCR product is amplified from a single target

Absolute Quantitative Real-time RT-PCR

• Construction of a standard curve: Construct a synthetic cRNA of your interest Reverse transcribe the cRNA into cDNA Set up Real-time PCR with known and increasing amount of

cDNA Plot Ct value vs. starting quantity of cDNA

Levels of Gene Expression Determined by Absolute RT-PCR

NKX2.5 GATA 5

Norm. GI,GII

β-actin

Norm. GI,GII Norm. GI,GII

Norm.

GI,GII

Norm.

GI,GII

Norm.

GI,GII

Reading Assignment: Chun et al., 2006. Trout Ea4- or human Eb-peptide of pro-IGF-I disrupts heart, red blood cell, and vasculature development in zebrafish embryos

Examples: Levels of Gene Expression Determined by Absolute RT-PCR

GATA 1 GATA 2 VEGF

Norm. GI,GII

Norm.

GI,GII

Norm. GI,GII

Norm.

GI,GII

Norm. GI,GII

Norm.

GI,GII

Reduction of mRNA Levels of Heart, Vasculature and Blood Cell Development-Related Genes in rtEa4-Peptide Injected Embryos

Gene

Molecules of mRNA/embryo at 36 hpf

NormalDefective embryos

Fold of reduction

NKX2.5 2.77±2.04X106 8.02±5.96X103 242.1±1.0

GATA5 3.94±1.07X105 2.41±6.58X104 11.7±0.9

GATA1 1.63±0.46X106 2.27±0.12X105 5.5±1.2

GATA2 1.37±0.22X104 3.59±0.76X103 2.7±1.0

VEGF 2.87±0.96X105 3.09±0.41X104 6.4±1.1

β-actin 5.77±1.19X106 4.16±1.33X106 1.0±1 .0

Comparative Quantitative RT-PCR

Fold =1/[2-(∆ ∆ CT)]

∆CT= CT (target) – CT (normalizer)

∆∆CT = ∆CT (treated) - ∆CT (control)

*Fold of reduction = 1/[2-(∆ ∆ CT)], where ∆ ∆ CT is ∆ CT sample - ∆CT control

Example: Levels of Gene Expression Determined by Relative Comparative Real-Time RT-PCR

Reading Assigned:

1. Chun et al., Molecular Reproduction and Development, 73: 1112-1121, 2006.

2. Chen et al., Journal of Cellular Biochemistry 101: 1316-1327, 2007.

Transgenic Mouse

• Transgenic mouse was first produced by Richard Palmiter and Ralf Brinster around 1980 by micro-injecting human growth hormone gene under the regulation of a metallothionein gene promoter into the pronucleus of mouse oocytes. The injected oocytes were implanted in the uterus of the pseudo-pregnant female mouse for further embryonic development

• Transgene: Human growth hormone gene driven by the promoter of metallothionein gene

• The transgenic mice grew much faster than the control littermates

• Palmiter et al. Nature 300:611, 1982

Production of Transgenic Mice

• This technique can be used to produce animal models with (i) a specific gene been knocked in, (ii) a specific gene been knocked out or (iii) a specific gene been down of its expression

Microinjecting a gene into the pronucleus of a fertilized egg

Inactivating a Gene by the Knock Out Technique

• The knock-out technique is essential for studying the function of a gene

• To conduct this experiment, an embryonic stem cell line is required

• neor: a marker gene that confers resistant to G-418

• tkHSV: a marker gene confers sensitivity to ganciclovir

• When the above construct is introduced into mouse stem cells, stem cells with gene been disrupted can be isolated

• The isolated ES cells can be used to generate gene knock out animals by the method described in next slide

Isolation of Recombinant Stem Cells with a Gene Been Knocked Out

Production of Germ Line Knock Out Animals

• Introduce the knock out ES cells into the blastoceal cavity of mouse embryos by microinjection

• Transfer embryos into pseudopregnant female mice

• Select chimeric mice• Mat chimeric mice with homozygous

black mice• Select brown mice for homozygous knock

out mice

What is Myostatin (MSTN) ?

• Originally identified as a growth differentiation factor 8 (GDF-8) in mice by McPherron et al. (1997)

• A member of the transforming growth factor-β (TGF-β) superfamily

Secreted factors that mediate key events in tissue growth and development through signal transduction cascades

Playing important roles in regulating embryonic development and in maintaining tissue homeostasis in adult animals

• Expressed initially in the myotome compartment of the developing somites

• Continuously expressed in the myogenic lineage throughout the development and in adult animals (Lee and McPherron, 2001; McPherron and Lee, 1997)

Myostatin (MSTN)• Encods 370 amino acid residues

• Contains all of the hallmarks of TGF-β superfamily peptidesA signal peptide for secretionA proteolytic processing site - RXXR9 cysteines in C-terminus and forming dimer by

disulfide linkage

RXXR

N

N

C

C

9 cysteines

Disulfide linkage

Proteolytic siteSignal peptide

Mature peptide

Pro-region

MSTN: A Negative Regulator of Skeletal Muscle Growth

• Mutation of MSTN gene in cattle leading to double-muscled phenotype (McPherron and Lee, 1997)

Belgian blue Piedmontese

Increased Skeletal Muscle Mass in MSTN-null Mice

(McPherron et al. Nature 387: 83-90,1997)

MSTN null mice Wild type

• Myostatin knockout mice exhibit a dramatic increase of skeletal muscle mass resulting from an increase of hyperplasia and hypertrophy of muscle cells

Effect of Knocking Out Myostatin Gene on Skeletal Muscle Development

Control

Knock- out

Knock Out Genes in Somatic Cells by the loxP-Cre Recombination System

• loxP site: site-specific DNA recombination site

• Cre: enzyme catalyzing recombina-tion (recombinase)

• Cre gene expression is driven by a tissue-specific or developmental-stage specific promoter

Micro RNA (miRNA)

• Micro RNA: was discovered in nematode (C. elegans) during analysis of

lin-4 and let-7 genes Cloning and nucleotide sequence analysis revealed that lin-4

and let-7 do not encode any protein product, but encoding RNAs of 21 and 22 nucleotides long, respectively. While lin-4 RNA hybridizes to the 3’-untranslated region of lin-14 mRNA and degrades the mRNA, let-7 RNA hybridizes to the lin-28 mRNA by the same manner and degrades the mRNA

About 100 different miRNA have been found in C. elegans and at least as many found in human. These miRNAs are used to degrade many mRNAs

All miRNA precursors are about 70 nucleotides long, and are processed by a ribonuclease (Dicer) to produce mature miRNAs of 21-22 bases

Base pairing between the miRNA and the 3’-untranslated region of an mRNA does not have to be 100% complementary. This differentiates it from the RNA interference

Interference RNA (RNAi or siRNA)

• RNA interference: RNA interference induces degradation of mRNAs with sequence

complementary to double-stranded RNAs RNA interference was discovered from initial attempts to inhibit

the translation of an mRNA by microinjecting a RNA with complementary sequence (antisense inhibition). In the control experiment, scientists found that perfectly base-paired double-stranded RNA of a few hundred pairs long was much more effective in inhibiting the expression of the gene than the anti-sense RNA alone

It was found that as long as one of the strand of the ds-RNA is complementary to the mRNA sequence, it can effectively inhibit the mRNA

Further studies revealed that the long double-stranded interfering RNA was processed into short dsRNA (siRNA) of 21-23 nucleotides double stranded region with 3’ single stranded and Dicer ribonuclease is required for the processing

Knock-down a Gene by Using RNAi

• In vitro or in vivo production of siRNA

• Inhibition of mex3 mRNA by injecting siRNA into the cells

Expression of mex3 gene in C. elegans embryo

Reading Assignment:1.RNA Interference2.Nobel lecture by Mello on RNA interference

• Microarray: a technique that allows you to determine the expression of many genes at one time

Reading Assignment: DNA microarray

.

• Genes (cDNA or oligonucleotides are spotted on glass plates to make gene chip

• Messenger RNA is reverse transcribed into cDNA and labelled with Cy3 (emission 570nm, green) or Cy5 (emission 670 nm, red)

• Hybridization (mixing Cy3-cDNA and Cy5 cDNA)

• Scan the slide to detect the hybridization signals

Principle of DNA Microarray

Enlarged Photo of a Microarray Chip

• This array has 2400 human genes

• Red indicates increase of expression; yellow equal expression and green reduce of expression

• This technique can help to determine the profiles of gene expression

Interpreting the Scanned Image

• The measured intensities from the two fluorescent reporters have been false-colored red and green and overlaid

• Yellow spots have roughly equal amounts of bound cDNA from each cell population and so have equal intensity in the red and green channels (red + green = yellow)

• Spots whose mRNA’s are present at a higher level in one or the other cell population show up as predominantly red or green

• The ratio of fluorescent intensities for a spot is interpreted as the ratio of concentrations for its corresponding mRNA in the two cell populations

Why DNA Microarray Technology?

• Gene discovery: Examples Profiling of cancer-specific expressing genes Tissue-specific expression of genes Developmental-specific expression of genes

• Disease diagnosis: Collections of genes showing expression of genes specific

to certain types of diseases Examples: Specific cancer type, hematopoietic disease etc.

• Drug discovery: Pharmacogenomics To find correlations between therapeutic responses to drug

and gene profiles of patents

• Toxicological research: Toxicogenomics• To find correlations between toxic responses to toxicants

and chages in the gene profiles of the objects exposed to such toxicants

Example

Novel Anti-cancer Activity of Pro-IGF-I E-Peptide

Assigned Reading:Chen et al., 2007: Suppression of Growth and Cancer-Induced Angiogenesis of Aggressive Human Breast CancerCells (MDA-MB-231) on the Chorioallantoic Membraneof Developing Chicken Embryos by E-peptide of Pro-IGF-I

B C A DS E

Signal peptide

Mature IGF-I E-peptide

Insulin-Like Growth Factor (IGF)-I

Post-translational processing

EB C A DS

Primary translation product of IGF-I

**IGF-I: Insulin-like growth factor-I; IGF-II: Insulin-like growth factor-II

What Is E-Peptide of Pro-IGF-I?

• The pro-hormone peptide of IGF-I

• It contains about 77 amino acid residues

• Presents in fish, human and other animals

• It is highly soluble in aquaous buffers

• Can be readily prepared by expressing the cDNA of E-peptide in E. coli or other cell systems

• Exerts dose-dependent mitogenic activities in established non-transformed cell lines (Tian et al., 1999)

• Induces morphological differentiation and inhibits anchorage-independent growth in oncogenic transformed cell lines (Chen et al., 2002; Kuo and Chen, 2002)

• Inhibits tumor cell growth and invasion, and tumor-induced angiogenesis in developing chicken embryos (Chen et al., 2007)

• Induces programmed cell death in cancer cells (Chen et al., 2011)

• Up-regulate fironectin 1 and laminin receptor genes and down-regulate uPA, tPA and TIMP1 (Siri and Chen 2006a, 2006b)

Novel Anti-Cancer Activities of the E-Peptide

Is There Molecular Evidence

• If E-peptide of IGF-I can control cancer cell growth, there must be genes that are up- or down-regulated by E-peptide

• Using microarray analysis technique, we determined genes related to cancer cell activities that are up- or down regulated by E-peptide in aggressive human breast cancer cell (MBA-MD-231) Isolate RNA samples from MBA-MD-231 cells treated wit E-

peptide and untreated cells Prepare cDNA from both RNA samples Label E-peptide treated cDNA with Cy3 and the untreated

cDNA with Cy5 Combine both labeled cDNAs and hybridized to a high

density human gene chip and scan the results

Results of the Microarray Assay

• From repeat screening a gene chip containing 10,000 human genes, more than 1,000 genes were found to be up- or down-regulated by E-peptide to different degrees

• Those genes with ratio of treated/untreated signals over + or – 2 are considered real difference

• Examples of up-regulated and down-regulated genes are summarized in the next two slides

• To confirm the microarray results, the mRNA levels of each gene in the treated and untreated RNA samples should be determined by real-time quantitative or comparative RT-PCR

Some Genes Down-Regulated by E-Peptide

• Oncogenes: TC 21, P53, vav 1, v-myb, c-H-ras (p21), sarc, v-erb 2 (high expression in tumors)

• Angiogenesis: Cysteine-rich angiogenic inducer 61 IGF-II, IGF-IIR, IGFBP-3, -4 & -7

• Cell adhesion: Catenin -like-1, Integrin -3, Integrin -3, Integrin -4, Tight junction protein 1

• Proteases: Cathepsin Z, Plasminogen activator inhibitor type I, Urokinase plasminogen activator, Tissue plasminogen activator

• Cell cycle, growth & proliferation: Cyclin-independent kinase inhibitor 1A, Latent transforming growth factor -binding protein 3, Serine inducible kinase, Colony stimulating factor 1, Thymosin -4

• Cytoskeleton molecules: Keritin-7, -8, and -18

Some Genes Up-Regulated by E-Peptide

• Cell adhesion: Fibronectin 1, Laminin receptor 1

• Cytoskeleton molecules: Keratin-1, Restin

• Proteases and inhibitors: Tissue inhibitor of metalloprotease I, Cathepsin C, Zinc metalloprotease

• Cell cycle, growth & proliferation: Cyclin A2, B1, B2, Cyclin-dependent kinase 7, Topoisomerase II

• Cell signal transduction molecules: TyrO3 protein kinase, Mitogen-activated protein kinase kinase 3, c-sar tyrosine kinase, Phosphotidylinositol-4-phosphate- 5 kinase type I

• Tumor rejection antigen (gp96) 1, Cdc 14 phosphatase, Protein tyrosine kinases, Heat shock proteins 70 and 90

Comparative Real-Time PCR Analysis of Genes Up- and Down-Regulated by E-Peptide

Names of Genes Relative Expression Levels

uPA 0.52 + 0.10PAI 1 0.42 + 0.04BCL 2 0.28 + 0.04Cysteine-rich angiogenesis inducer 0.86 + 0.12Tumor-associated Ca++ signal inducer 0.44 + 0.12TYPO3 protein tyrosine kinase 3.02 + 0.80Tumor rejection antigen (Gp96) 3.46 + 0.96Heat shock protein 90 KDa protein 13.40 + 0.35Heat shock 70 KDa pr5otein 10 2.92 + 0.39Capase 3 4.58 + 0.35Fibronectin 1 2.32 + 0.31Laminin receptor 1 1.41 + 0.11

RNA-Seq TechnologyRNA-seq, also called "Whole Transcriptome Shotgun Sequencing" ("WTSS"), refers to the use of high-throughput technologies to sequence cDNA or RNA in order to get information about a sample's RNA content. The technique has been adopted in studies of diseases like cancers. With deep coverage and base-level resolution, this technology provides information on differential expression of genes, including gene alleles and differently spliced transcripts; non-coding RNAs; post-transcriptional mutations or editing; and gene fusions

Reading Assignment:

1.RNA-Seq2.Ferrarini et al., 2012. application of the whole-transcriptome shotgun sequencing approach to the study of Philadelphia-positive acute lymphoblastic leukemia.

Chromatin Immunoprecipitation (ChIP) Assay

• ChIP assay can be used to detect the interaction of gene(s) with the bioregulator-receptor complex at the DNA level in the nucleus

• Examples: detecting the binding of steroid hormone or thyroid hormone receptors with other transcription factors in the promoter regions of genes regulated by these bioregulators

• It requires specific antibodies to bioregulator-receptor complex or transcription factors

Proteomics• Proteomics is the large-scale study of proteins, particularly their

structures and functions

• After genomics and transcriptomics, proteomics is considered the next step in the study of biological systems. It is much more complicated than genomics mostly because while an organism's genome is more or less constant, the proteome differs from cell to cell and from time to time.

• Unlike mRNAs, proteins can be modified via phosphylation, ubqitination, methylation, acetylation, glycosylation, oxidation and nitrosylation in the cell and resulted in a very comlicated picture. Therefore, studying the stuctures and functions of proteins in the cell may reveal a more realistic picture about the cell

• The proteome of a cell is studied by: Separating individual proteins by 2D-PAGE or other separation

methods Recovering individual protein from the 2D-gel Indifying the individual protein by mass spectrometry Computer analysis of the data

A 2D Gel

Comparison of Two Protein Samples

Strategy of Sequencing a Protein by Mass-Spectrometry

This analysis has enabled the establishment of protein database . The database facilitates the characterization of unknown proteins

Reading Assingment:

1. DNA microarray2. RNA-Seq3. Wang et al., 2009. RNA-seq: a revolutionary tool for

transcriptomics . Nature Review: Genetics 10: 57-63.4. Proteomics

Documents

II. Methods of Studying Bioregulation The Scientific Methods: Controls; Reprensentative sampling; Dose-response relationship; Biological rhythms Methods