User:Z3272212

From CellBiology

Image

HeLa cells treated with control siRNA, expressing Atg16L1-GFP (green) and incubated with Alexa555-CTX (red).jpeg

lab attendance

lab 1

--Z3272212 15:13, 8 March 2012 (EST)

lab 2

  • refer history

lab 3

  • refer history

lab 4

--Z3272212 14:18, 29 March 2012 (EST)

lab 5

--Z3272212 14:13, 5 April 2012 (EST)

lab 6

--Z3272212 14:10, 19 April 2012 (EST)

lab 9

--Z3272212 14:22, 10 May 2012 (EST)

LAB 10

--Z3272212 14:33, 17 May 2012 (EST)

lab 11

--Z3272212 15:30, 24 May 2012 (EST)

lab 12

--Z3272212 15:40, 31 May 2012 (EST)

lab 1

link

LECTURE 2

http://cellbiology.med.unsw.edu.au/cellbiology/index.php?title=ANAT3231_References

http://jcb.rupress.org/content/196/4/483

LECTURE 2


lab 2 microscopy

  • lights, fluorescent lights could damage the cell by generating free radicals. therefore minimise the light as much as possible.
  • tirfm - look at junctions on membranes? illuminates the region in contact with the glass. use in cell migration observation
  • time lapse - cell need to be maintained in environmental similar to its origins (37C, CO2),take a photo, shut down the light (low light exposure)
  • laser capture - laser cuts the area of interest, isolate a cell/group of cell. compare gene expression of normal and neoplastic cell/tissue.
  • 3d image (xyz axis) - confocal laser/STM
  • superresolution microscopy (NEW!)

lab 3

  • fixation technic
    • Fresh Frozen
    • Precipitation
    • Aldehyde Cross-linked

lab 4

  • 2 binding sites on each antibody molecule (same epitope)-Fab region
  • single antibody can bind to 2 antigen
  • papain separates fab and fc
  • monoclonal antibody - produced by a single B cell x myeloma hybrid (hybridoma)
  • grow and multiply indefinitely
  • selecting B cell - how?
  • anti igg antibody /anti-antibody

exercise lab 4

--Z3272212 14:56, 29 March 2012 (EST)

  • musashi-1 [1]
    • an evolutionarily conserved family of RNA-binding proteins that is preferentially expressed in the nervous system
  • musashi antibody [1] . pdf version [2]
    • rabbit igg
    • Immunohistochemistry: 1:100-1,000 Immunocytochemistry 1:100 Western Blot 1:100 ; dilution ratio
    • anti musashi-1 antibody [3]

lab 5

  • cell knockout method
    • transgenic - random chromosomal integration of foreign dna
    • creation of transgenic mice - take a fertilised egg, hold into a pipette, inject dna into male pronucleus, transfer to oviducts of pseudopregnant female. transgene present in all nucleated cells.
    • types of transgenic
      • target protein overexpression (eg GH transgenic)
      • target protein maybe ectopically expressed
      • mutated protein is expressed to produce: constitutively active (gain of function) or dominant negative (loss of function)form of protein or to mimic a mutated protein observed in a human genetic disease
      • the protein needs promotor to attach a mutant protein causes
    • due to randomness, each resultant founder contains the transgene in different site of genome
    • and the position effect can profoundly affect the expression of both the transgene and the endogenous genes whose regulatory elements may be disrupted
    • and transgene may disrupt the coding region
    • it is essential that lines from several different founder lines be examined before a conclusion relating a specific phenotype to transgene expression is made
    • to assess dose-response relationships between transgene expression and phenotype, it is also important to assess lines...
    • homologous recombination - site directed disruption (k/o) or replacement with a modified variant of a gene allele (knock-in). conditional KO/Ki - tissue specific and inducible
    • ko/ki mice - mouse in which a specific mouse gene has been genetically modified and the modification is transmitted through the germ-line
    • ko is a modification in which the activity of the gene is eliminated
    • ki is a modification in which a gene is introduced in
    • ko/ki - understand function of genes
    • study pathophysiology or test therapeutic approach
    • mimic recessive disorders
    • traditional transgenic can be used for dominant disorders
    • how to make ko mouse - principle: homologous recombination. fragment of genomic DNA is introduced into a mammalian cell
    • 1. pluripotent (undifferentiated) embryonic stem (ES) cells 2.culture these cells in vitro 3. gene targeting strategy: a. culture in vitro b. electroporate with targeting vector c. select and screen for targeted cell d. foster mother incubation
    • 1. es cells isolated from blastocyst 3.5dps 2. dna targeting construct into ES cells 3. microinjection microscope
    • positive selection vs negative selection
    • screening ES colonies
    • advance : conditional ko


  • gene function
  • function of gene product in adults
  • genetic diseases
  • cytoskeleton
    • microfilaments
    • intermediate filaments
    • microtubules
    • actin filaments : severing, nucleation, monomers, bundling, motor, side -binding (tropomysin), capping, cross-linking
    • tropomysin - filamentous,forms dimers, dimers interact head to tail to form polymer, interact along the length of actin, provide stability and regulate binding
    • different Tm isoforms regulate actin filaments by recruiting different actin binding protein
    • Tm5NM1/2 ko - eliminated in all tissue
    • Tm5NM1/2 transgenic (Tg) - overexpressed in all tissue
    • body weight, histopathology, food intake, food utilisation - unaffected
    • organ weight - Decrease in
  • summary: gene targeting used to delete or mutate an existing gene. Tg mice are use to study overexpression of gene product.

lab 6

Cell biology lab 6.JPG

  • Genotype A is Tm4 and Genotype B is the control.
  • Genotype A expressed more stringed cells than B but B express more broken fan than A.

task 7

contribution to group project:

  • task delegation
  • introduction of p53 pathway
  • currently working on the normal function and abnormality of the p53 pathway

final contribution:

  • added 'estrogen' section under 'protein' section
  • uploaded a computer version of a group member's original hand-drawn image, making it more visible
  • added the 'Li-Fraumeni Syndrome' under the 'abnormality' section
  • added some info under 'nanoparticle mediated p53 therapy' in the 'current research' section


--Z3272212 13:58, 3 May 2012 (EST)

task lab 8

1. Identify a mammalian cell line in the ATCC catalogue (and add a link)

  • ATCC -DYP0530 Human Induced Pluripotent Stem Cells ATCC

2. Identify the original tissue of origin of that cell line.

  • Organism: Homo sapiens
  • Age: 63 years
  • Gender: Male
  • Ethnicity: Caucasian
  • Source: Cell Type: Human iPSC

3. Identify the original paper that characterised the properties of that cell line.

In the pubmed search, the term used was ACS-1014 and revealed a few paper related to the search term. This paper is about PARP inhibitor in breast cancer [2]

LAB 10

- Tissues that had been trialed for Stem Cell transplant: Bone marrow, skin, Skeletal muscle, liver, brain or spinal cord, heart

- Problem for transplant: Migration, route of delivery, rejection, specific cell type and amount needed to be administered

- Advantage of treatment with chemotherapy agent: efficient elimination of endogenous stem cell, create receptive niche for deposition of donor cell

- Tiger snake venom triggers muscle regen when introduced

- Inject chemotherapy - resistant muscle stem cell plus ??

- isolate stem cell from satellite cells

- cd34+ cells from skeletal muscle; heteregoneous mix of cell type containing hematopo & muscle sc, easy to isolate using immunomagnetic beads; damage muscle,activate muscle, activate stem cell,

- how to differentiate host and donor cell? male donor, female recipient.

- how to quantify engraftment? PCR.

- 3 days prior: activate muscle regen in male donor and add stem cell one day before extraction

- extract donor stem cell

- activate muscle regen in recipient (female), inject donor stem cell

- day 4 tissue collection: y-chromosome q-pcr/fish.

- enhanced engraftment: conclusion; BCNU/O6BG prevent regen in muscle, MGMT (P140K) expressing cell engraft in muscle in the presence of BCNU but regenration driven by MGMT expresing cells recruits endogenous cells.

Lab 9

--Mark Hill 13:47, 17 May 2012 (EST) You have not completed the peer assessment process yet. If you have made comments on each project page they need also to be pasted here today for me to include in your individual assessment.

Lab 12/Final Lab

1. Identify a current technique used in gene sequencing

  • Next generation Sequencing
    • The Ramaciotti Centre now has the technology apparatus to perform NGS such as The Illumina HiSeq 2000 system and MiSeq Ramaciotti
    • The NGS allows more output and hence faster sequencing process. Not only DNA, but RNA can also be sequenced[3]


2. Identify a recent cell biology research paper that has used microarray technology?

  • "A systems biology approach reveals common metastatic pathways in osteosarcoma" - a study by Flores et al published very recently this year (May 2012) that integrated genomic and proteomic data to understand the mechanism of metastasis in osteosarcoma. [4]

3. What aspect of the research findings were contributed by the microarray technique?

  • mRNA expression microarray analysis together with glycoproteomic analysis were performed on human metastatic osteosarcoma cell line, particularly HOS/143B and SaOS-2/LM7. These methods allow for the pathway analysis of the metastatic cell line. The researchers came up with Cytoskeleton remodeling/TGF/WNT pathway [5]

References

  1. Hideyuki Okano, Hironori Kawahara, Masako Toriya, Keio Nakao, Shinsuke Shibata, Takao Imai Function of RNA-binding protein Musashi-1 in stem cells. Exp. Cell Res.: 2005, 306(2);349-56 PubMed 15925591