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Manipulating Genes

Posted by star on 2019-05-16 02:02:17
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Gene isolation, modification and amplification have been described in previous section. An approach called transfection make it possible to introduce gene of interest into cultured cells, where they can be transcribed and translated, This approach can be used to increase the concentration of molecule of interest and detect its function in cells, There are various methods of transfection: some rely on physical treatment (electroporation, nanoparticles, magnetofection), other on chemical materials (lipofection, calcium phosphate, DEAE-dextran) or bio-logical particles (viruses) that are used as carriers. Then the transfected cells is cultured in particular medium in which nontransfected cells cannot survive, thus, those cells that have been inserted the foreign DNA can be selected.
Through transfection, the DNA fragment carrying a gene of interest usually inserts into the chromosomes randomly, it can replace one of the two copies of the normal gene by homologous recombination. If the gene replacement happens in the germ-line cells, it can then be passed on to progeny. These animals which chromosome permanently altered and contain the foreign or modified DNA are called transgenic animals. For example, the target gene can be replaced in the transgenic mouse. Transgenic animal has presented excellent model for understand gene function. If both copies of the target gene is deleted or completely inactivated, the resulting animal is called knockout animal. Any DNA sequence or gene within the genome can be altered using this method.

Gene knock out technique is generally used to prove Gene knock out for a special process. However, sometimes the deletion of the target gene has little or no alteration in the animal’s phenotype. It is difficult to interpret such results. In some cases, the abs......

Cell Isolation and Cell Culture

Posted by star on 2019-05-15 00:55:33
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Regardless of the species to be studied, growing large populations of isolated cells for biochemical analysis and microscopic observation is helpful.This is straightforward for the unicellular organisms such as fungi or bacteria, which can be grown suspended in a nutrient medium. These organisms can also be grown on the surface of gelled agar in a petri dish.When single cells are dispersed widely on an agar surface,each multiplies to form a macroscopic colony, all descendents of a single cell. This family of cells is called a clone.
For multicellular organisms, it is often possible to isolate single live cells by dissociating a tissue with proteolytic enzymes and media that weaken adhesions between the cells.Tissues provide the most realistic source of material. Several approaches are used to separate the different cell types from solid tissue or liquid tissue. For solid tissue, the first step in isolating individual cells is to get mixed cell suspension through proteolytic enzymes digestion to disrupt the extracellular matrix and cell-cell junctions. After that, a flow cytometer, also known as fluorescence activated cell sorter (FACs), can be used to isolate target cells from the mixed cell suspension. For isolating cells, an antibody coupled to a fluorescent dye is used to specifically bind to the surface of specific cells, then the labeled cells are chosen from the unlabeled ones using the flow cytometer. 'The flow cytometer can sort 20 thousand cells each second, the purity of selected cell can reach to 95%. Immunomagnetic separation(lMS) is another useful tool that can efficiently isolate viable and functional cells, ln one approach, cells are incubated with immunomagnetic beads precoating antibodies which will bind to antigens present on the surface of cells, thus these bead-attached cells will be captured by a magnet placed on the side of the test tube. Compared with FACs, this technique is easy to work and its procedure is very rapid. In addition, a ......

Observing Plant Cytoskeleton under Optical Microscope

Posted by star on 2019-05-12 19:16:53
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5.1 Objectives
1. Learn the slide preparation method for cytoskeleton.
2. Learn to stain the plant cytoskeleton by the method of Coomassie bright blue R250.
3. Observe the morphology of cytoskeleton by optical microscope.
5.2 Introductions
Plant cells have highly dynamic cytoskeletal networks including microtubules and actin
microfilaments involving in plant growth and development. The cytoskeleton of human and animal cells has the same two components of microtubules, actin microfilaments as those of plant cells and the unique intermediate filaments involving in the keeping of cell morphology, cell movement, material transport, energy conversion, signal transduction.
5.3 Principles
The proteins of cytoskeleton are inside the cell and can be observed under optical microscope after increasing the permeability of cell membrane by detergent Triton X-100 treatment and staining with Coomassie bright blue R250.
5.4 Apparatuses, materials and reagents
Apparatuses: optical microscope, dropper.
Materials: onion, slides, lens paper, pH meter/test paper, tissue.
Reagents:
1.0.01 mol/L phosphate physiological buffered saline (PBS)
2.1%Triton X-100/M-buffer
3.3%glutaraldehyde PB solution
4.0.2% Coomassie bright blue R250

5.5 Protocols

Procedure:

1. Peel off the onion inner membrane at the size of l cm by forceps.

2. Flat the membrane in PBS buffer and keep it wet.

3. Take it out, place it on glass slide and make it dry by absorbing the extra buffer.

4. Add 2 drops of 1%Triton X-100/M buffer onto the slide and keep it for 5 mins to increase membrane permeability.

5. Dry off the buffer and fix the slide by 3%glutaraldehyde PB solution for 30 mins.

6. Wash with PBS buffer twice.

7. Stain by 0.2% Coomassie bright blue R250 for 30mins.

8. Wash with PBS buffer twice and dry it by tissue.

9. Observe under optical micro......

Observing Several Organelles under Optical Microscope

Posted by star on 2019-05-12 19:12:45
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4.1 Objectives
1. Learn to identify the organelles of mitochondria, Golgi complex, Nissl bodies (endoplasmic reticulum) and centrosome under optical microscope and familiarize with the shape and distribution of organelles under optical microscope.
2. Master the method for the usage of microscope,

4.2 Introductions
In cell biology, cell organelles are specialized subunits inside the plasma membrane and have their specific functions. The organelles of a eukaryocytic cell are usually enclosed within their own lipid bilayers in the cytosol. They are the basic structural and functional units of a cell including mitochondrion, Golgi apparatus, chloroplast, endoplasmic reticulum, centrosome etc.

Mitochondrion is a double-membrane compartment ranging from 0.5 to I.0 micrometer (μm) in diameter. The compartment includes outer membrane, intermembrane space, inner membrane, and cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Mitochondria are described as cellular energy plants, because they generate most of the cell's supply of adenosine triphosphate (ATP) from the oxidation of glucose substances.
Golgi apparatus, also known as Golgi complex, is single-membrane compartment in all eukaryotes. It was identified in 1897 by the Italian physician Camillo Golgi and named after him in 1898. Its function is protein sorting, packaging, processing and modification.
Chloroplast is double membrane compartment in plants cell and Protista. lt traps energy from sunlight by photosynthesis.
Endoplasmic reticulum (ER for short) is single membrane compartment in most types of eukaryotie cells, including the most primitive Giardia, but is absent red blood cells and spermatozoa. It has an interconnected network of flattened, membrane-enclosed sacs or tubes known as cisternae. The membranes of t......

Breast cancer resistance may be related to a diet rich in leucine

Posted by star on 2019-05-12 19:01:15
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About one in ten American women will develop breast cancer in her lifetime. Most breast cancers rely on estrogen for growth. Estrogen-receptor-positive breast cancer is often treated with the drug tamoxifen, which blocks the hormone's effect on the tumor. However, many tumors eventually become resistant to tamoxifen, leading to cancer recurrence or metastasis.
Now, a team of researchers at duke university’s cancer institute has found a possible link between leucine levels and tamoxifen resistance in ER+ breast cancer. At the same time, the researchers further identified a key protein that can introduce leucine into cells and modulate the sensitivity of ER+ breast cancer cells to tamoxifen, revealing a mechanism for overcoming endocrine drug resistance in ER+ breast cancer patients.
"The survival of ER+ breast cancer patients with drug-resistant and metastatic tumors is very short, usually less than three years, because their treatment options are very limited," says Helen piwnica-worms, Ph.D., director of the cell biology program at the BIDMC cancer institute." Our findings in the laboratory suggest that lowering leucine levels inhibits tumor cell proliferation, while increasing leucine levels enhances cancer cell proliferation." These findings also provide evidence for the possible benefits of a low-leucine diet.
Leucine is one of the 20 amino acids that make up all proteins in the body. It is also one of the nine essential amino acids that must be obtained through food. Beef, chicken, pork and fish are rich in leucine. Because cells by themselves don't produce leucine, Helen piwnica-worms and his colleagues were able to test how controlling leucine levels can affect the growth of human-derived ER+ breast cancer cells. Lowering leucine levels inhibited division in ER+ breast cancer cells, while increasing the number of amino acids by a factor of 10 increased the division, the researchers said.

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