Animal Sciences Internship: Student Research Assistant

1
Animal Sciences Internship Student Research
Assistant

• Kristen Meiers
• Summer 2005

2
Background Information

• Dr. Kichoon Lee
• Assistant Professor- Animal Sciences Dept.
• 
  Disciplines
• 
  Growth and Development
• 
  Genetics
• 
  Nutrition
• 
  Physiology
• 
  Species
• 
  Beef, Poultry, Sheep, Swine,
• 
  Rodents

3
Area of Research Molecular Biology

• Identifying and defining genetic and metabolic
  networks that will promote the efficiency of
  livestock production
• The effect of target genes on fat and muscle
  development
• Food intake regulation

4
Area of Research Molecular Biology

• Evaluating target genes that are involved in
  mechanisms of metabolic diseases
• Treatment of human metabolic diseases
• Identifying molecular mechanisms concerning food
  intake regulation
• Example glucose regulation

5
Application

• Identifying the genes that control the mechanisms
  allowing muscle cells to differentiate into fat
  cells
• Animal Sciences marbling in meat
• Humans Obesity
• Type 2 diabetes

6
Area of Research Molecular Biology

• Discovering genes through
• Microarray- computerized system to screen
  several thousand genes at one time
• cDNA Arrays Single-stranded DNA that is
  complimentary to mRNA in the presence of reverse
  transcriptase
• Oligonucleotide Chips Relatively short
  single-stranded nucleic acid chain ( 20-70
  nucleotides)

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cDNA or Oligonucleotide Array Experiments
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Spotted
Microarray
Human 10k Array
8x4
subarrays
Total 324 spots
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Microarray and Functional Genomics for Fat
Metabolism and Obesity Studies

• Microarray High through-put screening
• If gene is known Study the function of the gene
  in adipocyte development
• If the gene is not known Cloning of the gene by
  RT-PCR and characterizing the gene using
  bioformatics, such as gene banks, protein
  homology, or a domain search

10
Microarray and Functional Genomics for Fat
Metabolism and Obesity Studies

• Functional Genomics
• In- vitro system using 3T3-L1 pre-adipocyte,
  looking at over expression and under expression
  of the target gene
• In-vivo approaches using transgenic mice or
  knock-out mouse models
• Data base search of any gene located at a
  possible human genetic obesity loci

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Area of Research Molecular Biology

• Finding functions and roles of genes through
• In-Vitro- cell cultures
• In-Vivo- transgenic and knock-out mouse models

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In-Vitro Systems

• Artificial environment outside of a living
  environment
• Multiplying, Identifying, and cloning of a gene
  in a bacterial culture system

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In-Vivo Systems

• Within a living organism
• Collect tissue samples from mice
• Heart
• Liver
• Kidney
• Brain, etc.
• Isolate RNA from each tissue

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My Job as a Research Assistant

• Primarily aid Dr. Lees post-doctoral and Phd
  candidate with specific lab procedures
• RNA isolation
• Standard Gene Cloning
• Preparing and Running
• agarose gels
• PCR
• Preparing Buffers, broths, agar
• Digestion of samples with restriction enzymes

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My Job as a Research Assistant

• Daily upkeep of lab
• Sterilizing equipment
• Immunohistochemistry
• Ordering supplies
• Organizing records

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Gel Electrophoresis

• Purpose Separates DNA fragments according to
• size
• Electric current is used to move DNA across the
  polysaccharide gel
• Smaller molecules are
• able to navigate gel
• faster than larger
• molecules

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Gel Electrophoresis

• Dilute agrose powder in TAE solution
• Microwave to dissolve agarose
• Add Ethidium Bromide
• Pour gel in tray- let solidify
• Load samples in appropriate wells and run
• Take picture of gel

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RNA Isolation

• Purpose Compare expression levels of genes
• Procedure
• Lyse cells from culture with Trizol
• Add chloroform- vortex- centrifuge
• Transfer aqueous phase and add isopropyl alcohol-
  vortex

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RNA Isolation

• Incubate at room temperature- centrifuge
• Pellet forms- Remove supernatant liquid
• Wash with ethanol- centrifuge
• Remove ethanol- let pellet dry
• Suspend in RNase free water and Formamide
• Store at -80C
• gtConfirm quality and quantity of bends with
  electrophoresis ( 2 clear bends)

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Mini-Prep Standard Gene Cloning

• Purpose Isolation of plasmid containing a
  target gene with the correct orientation
• Procedure
• Obtain broth cultures from shaker (incubated at
  37C)
• Transfer to microtube- centrifuge to get pellet-
  dump out broth

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Mini-Prep Standard Gene Cloning

• 3. Add Buffer P1 to degrade the RNA- vortex
• 4. Add Buffer P2 to lyse the E.coli- invert
• 5. Add Buffer N3 to separate protein out- invert
• 6. Centrifuge
• 7. Remove supernatant- DNA
• 8. Wash with PE Buffer-centrifuge, extract with
  water

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Mini-Prep Standard Gene Cloning
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Mini-Prep Standard Gene Cloning

• Now the sample can be used with a particular
  restriction enzyme in a reaction
• Using gel electrophoresis you will be able to
  tell if the gene cloned displays the correct size
  and orientation of base pairs

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• Microarray System (screen 12,000 genes)

Isolation of gene by RT-PCR (10 genes)
Clone more genes with cDNA (Mini-Prep)
Conformation of gene expression by quantitative
RT-PCR (Isolate RNA)
Further functional Study
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Findings

• Unknown gene that is highly expressed in fat and
  muscle

60
50
cyc
40
30
target gene /
20
10
Isolate RNA and real-time-RT-PCR for
quantification
0
fat
fat
lung
liver
brain
muscle
kidney
heart
spleen
testes
BAT
intestine
epi
ing
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Findings

• Fat contains two different fractions
• Stromal vascular fraction non-fat cell and is
  mostly pre-adipocyte cells
• Fat fraction
• Gene highly expressed in fat fraction

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Fat
SV
Fraction
Target gene
dlk1
cyc
Fat fraction Stromal vascular fraction
RNA isolation ?RT-PCR
Fat tissue
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Findings

• In mouse 3T3-L1 pre-adipocyte start expressing
  highly between days 4 and 8

40
40
cyc
cyc
30
30
20
20
target gene /
target gene /
10
10
0
0
0
2
4
6
8
0
2
4
6
8
Oil
-
Red
-
O (day 8)
Oil
-
Red
-
O (day 8)
Day of 3T3
-
L1 cell differentiation
-
L1 cell differentiation
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Summary

• Target gene is highly expressed in adipose tissue
• Expression levels increase during fat cell
  development
• Continue with further studying to know function
  of gene

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My Experiences

• Dont be afraid to try something new!
• Better understood how research is conducted and
  how much time and energy is put into it
• Became much more familiar with reading scientific
  papers
• Learned so much about biological concepts
• Enhanced my lab techniques
• Became better at multi-tasking and managing time