Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency. Other mechanical methods of introducing a gene of interest, such as a �gene gun� or microinjection, are also limited by poor cell viability and low numbers of transfected cells. The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns. We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells. Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection. The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Preparation

1. Make sure that there are plenty of cells available for transfection (several million cells for each DNA to be transfected).
2. Coat a tissue culture dish, into which the transfected cells will be seeded, with 10 µg/ml human fibronectin the night before transfection. Right before starting the transfection protocol, remove the fibronectin, rinse the dish with PBS, and place culture media in the dish. Leave the dish in the 37°C tissue culture incubator to warm the media. Also, place 1 ml of culture media in the incubator to pre-warm to 37°C.
3. Place the DNA(s) to be transfected, and transfection solution (from Amaxa), on ice.

Transfection

1. Wash a defined number of cells with PBS (we usually use ~ 5 x 106 cells per transfection). Pellet the cells by centrifugation (1000 rpm (~200xg) for 5 minutes), remove as much PBS as possible, and resuspend the cell pellet in 100 µl of the transfection solution provided with the transfection kit. Transfer the cell suspension to a 1.7 ml eppendorf tube.
2. Add 5 µl of the DNA (~5-10 µg of DNA per transfection) to the cell suspension and mix gently.
3. Using the Amaxa mini-pipette, transfer the cell-DNA mix into the Amaxa transfection cuvette. Make sure you have 1 ml of pre-warmed culture media for the next step. Also, remove the cell dish with culture media from the incubator and place it in the tissue culture hood.
4. Place the cuvette into the Nucleofector, rotate the wheel clockwise, and select program "A-033". Press Enter.
5. A successful transfection is indicated by the presence of a very dense layer of bubbles on top of the solution in the cuvette. Add 500 µl warm media into the cuvette, and using the Amaxa mini-pipette, transfer the transfected cells into the dish with pre-warmed media. Rinse the cuvette with more warm media and add the rinse to the same dish. Place the dish with cells in the 37°C tissue culture incubator.

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Autism and FMR1 Gene Mutation Lines now Available

Through the National Human Neural Stem Cell Resource (NHNSCR), we have created an Autism induced pluripotent stem cell (iPSC) Biorepository (ASCB) and now have available dozens of fibroblast lines taken from patients with idiopathic autism as well as patients with FMR1 gene mutations (both full- and pre-mutations), with and without an accompanying autism spectrum disorder. (iPSC) lines from many of these fibroblast cultures are also available for distribution; neural stem cell derivatives will soon follow. More information on these lines will be posted soon.
All of the cultures are established under an NIH grant (R01HD059967), entitled “An Open Repository of Autism iPSCs and their derivatives”. In addition to acting as a bank for autism lines, the NHNSCR’s ASCB also acts as a training facility, bringing in scientists who wish to use these new cell lines in their research and giving them the essential skills to do so (see Figure). Finally, the ASCB is conducting a large bioinfomatic analysis of the whole genome expression data derived from these lines and is actively collaborating with other groups wishing to use these lines.
For more information, please e-mail Dr. Phil Schwartz at pschwartz@choc.org .

Standard Protocol for Immunohistochemistry

Materials Required
1. Slides
2. Phosphate buffered saline (pH 7.6)
3. Hydrogen peroxide
4. Primary antibody
5. Blocking serum (normal serum)
6. Biotinylated secondary antibody
7. ABC reagent (6, 7, and 8 are included in Vectastain Elite ABC kit)
8. Diaminobenzidine
9. Meyer's hematoxylin
10.Permount

Method
1. Incubate in a dry oven at 62oC for 1 hour. Slides should be maintained in a vertical orientation
to allow complete removal of the paraffin.
2. Dewax slides in xylene for 5 x 4 minutes.
3. Hydrate slides in 100%, 95%, and 75% ethanol for 2 x 3 minutes each.
4. Immerse slides in tap water for 5 minutes
5. Antigen retrieval method (optional).
6. Quenching of endogenous peroxidase (optional)
a.immerse slides in 3% hydrogen peroxide solution for 6 minutes.
b.wash slides in PBS for 3 x 5 minutes.
7. Incubate slides with blocking serum (1:50) for 30 min.*
8. Blot excess serum from section, and incubate with primary Ab. Suggested incubation time
(may vary between antibodies):
mAb 2 hours at room temperature or overnight at 4oC.
pAb: 1 ~ 1.5 hours at room temperature.
9. Wash slides in PBS for 3 x 5 minutes.
10.Incubate slides with biotin-conjugated secondary Ab for 30 min.*
11.Wash slides in PBS for 3 x 5 minutes.
12.Incubate slides with Avidin-Biotin Complexes for 30 min.*
13.Wash slides in PBS for 3 x 5 minutes.
14.Incubate slides in fresh DAB solution for 2 minutes. (We use DAB solution in Vector
DAB/Ni substrate kit).**
15.Stop the reaction by washing in tap water.
16.Counterstain in Meyer's hematoxylin for 10 seconds.
17.Dehydrates slides in 75%, 80%, 95% and 100% ethanol
18.Clear slides in xylene 4 X 5 minutes.
19.Mount cover slide with Permount.


* Blocking serum, secondary antibody and avidin-biotin-peroxidase complexes are included in most of the
immunostaining kit. Our lab uses the ABC kit from Vector Lab (Vectastain Elite ABC kit).
** We use DAB solution in Vector/DAB/Ni substrate kit (Vector Labs., Cat. SK-4100).

HOW IS MONOCLONAL ANTIBODY PRODUCED EXACTLY

Process by which large quantities of antibodies (targeted against a particular antigen X) can be produced.

A mouse is immunized by injection of an antigen X to stimulate the production of antibodies targeted against X. The antibody forming cells are isolated from the mouse's spleen.

Monoclonal antibodies are produced by fusing single antibody-forming cells to tumor cells grown in culture. The resulting cell is called a hybridoma.

Each hybridoma produces relatively large quantities of identical antibody molecules. By allowing the hybridoma to multiply in culture, it is possible to produce a population of cells, each of which produces identical antibody molecules. These antibodies are called "monoclonal antibodies" because they are produced by the identical offspring of a single, cloned antibody producing cell.

Once a monoclonal antibody is made, it can be used as a specific probe to track down and purify the specific protein that induced its formation

PLASMID EXTRACTION

Method
Many different methods are available for isolation of plasmid DNA. In this practical you will
be using a commercially available kit, which utilizes an affinity column to purify the plasmid
DNA. Bacteria (E. coli in this case) containing plasmid is grown from a single colony in a
suitable medium such as Luria Broth (LB). An aliquot of the culture is used for preparing
the plasmid DNA.

Centrifugation
Bacterial cult.Centrifugation results in pelleting of bacterial cells

Solution I

(Tris pH 8.0
EDTA- Ethylene diamine tetra acetic acid and
Glucose)
RNase
Lysozyme- optional

· Glucose gives osmotic shock that leads to the rupture
of cell wall and membrane
· EDTA, inhibits nucleases
· Rnase: Dergrades RNA
· Lysozyme is used for rupturing cell wall of bacteria.


Solution II
Sodium hydroxide
SDS- Sodium dodecyl sulphate
(Alkaline pH `12.0

Lyses the cell completely
· Alkaline pH denatures chromosomal DNA but not the
covalently closed circular plasmid DN

Solution III
Sodium or potassium acetate
(Acidic pH 5.4)

· Neutralizes the alkaline pH
· Precipitates protein and forms SDS-protein complex
· Chromosomal DNA renatures and aggregates with
protein

Centrifugation

· Pellets the Protein –DNA aggregates
· Plasmids will be present in the supernatant
(These plasmid can be precipitated with ethanol- This
used to be the conventional method)

Spin column
Silica membrane

· Silica membrane binds to plasmid DNA at high salt
buffer condition

Elution

· Under low salt condition (Distilled water), plasmids
can be eluted from the silica membrane

Protocol
Each group is provided with 10ml of an E.coli (containing plasmid) culture that has been grown
overnight at 37oC.
1. Transfer 1.5ml of bacterial culture to a micro centrifuge tube.
2. Pellet cells by centrifuging at 12,000 rpm for 2 minutes
3. CAREFULLY remove the supernatant.
4. Add another 1.5ml of culture to the same tube and centrifuge and repeat step 2 &3
5. Add 250 ml of Solution I /RNAase.
6. Resuspend the pellet by vortexing briefly or by pipetting up and down.
7. Add 250 ml of Solution II
8. Mix GENTLY by inverting and rotating the tube several times. DO NOT vortex!!!
9. Leave at room temperature for 5 minutes but NOT MORE than 5 minutes.
10. Add 350 ml of Solution III
11. Mix by inverting the tube 6-8 times
12. Centrifuge at 12,000 rpm for 12 min
13. While your tubes are spinning place a Spin column in a 2-ml collection tube.
14. After centrifugation, CAREFULLY transfer the supernatant to the column. DO NOT disturb the
pellet.
15. Centrifuge the column and collection tube at 12,000 rpm for 1 min
16. Discard the flow through collected in the collection tube.
17. Replace the column in the collection tube
18. Add 250 ml of HB Buffer into the column and centrifuge for 1 minute at 12,000 rpm and
discard the flow through.
19. Add 700 ml of wash buffer into the column and centrifuge for 1 minute at 12,000 rpm and
discard the flow through.
20. Centrifuge again to remove residual buffer for 90 seconds
21. Place the column in a sterile 1.5ml tube.
22. Add 50ml of deionised water or TE buffer into the column
23. Centrifuge at 12,000 rpm for 1 min.
24. Discard the column. Plasmid DNA is in the deionised water or TE buffer will be collected in the
1.5ml tube.
25. Place the tube containing plasmid DNA on ice for further use or store at –20oC

DNA ISOLATION FOR PLASMID

DNA ISOLATION FOR PLASMID ISOLATION

Reagents and solutions

1. Alkaline Solution I
50 mM glucose
25 mM Tris HCl (pH 8.0)
10 mM EDTA (pH 8.0)

2. 10 mg/ml lysozyme

3. Alkaline Solution II
0.2 N NaOH (2.0 ml of 10 N NaOH in 100ml double distilled water)
1.0% SDS

4. Alkaline Solution III
5 M Potassium acetate
Glacial acetic acid
Autoclaved water

5. TE buffer
10 mM Tris HCl (pH 8.0)
1.0 mM EDTA (pH 8.0)

6. Ring stand with clamp

7. 26 gauge needle

8. 1 cc syringe

9. Dialysis buffer
1 M Tris HCl (pH 7.5)
0.5 M EDTA (pH 8.0)
Autoclaved distilled water (1L)

10. Ampilcillin 100 µg/ml

11. Ice cold Isopropyl alcohol

12. Cesium chloride as per Plasmid DNA sample

13. Dialysis tubings (Nylon)

14. 5 M NaCl
29.22 g NaCl in 75 ml water and bring the volume to 100 ml.

15. Isoamyl alcohol

16. Luria Broth media
1 gm Tryptone
0.5 gm Yeast extract
1 gm Sodium chloride
Deionised water 75 ml
Shake all the solutes until dissolved. Adjust the pH to 7.0 with 5 N NaOH. Bring the volume of the solution to 100 ml with deionised water and sterilize it by autoclaving it for 20 minutes at 15 psi.

Procedure

Isolation of Plasmid DNA

1. Pick up a colony of bacteria and inoculate it in a conical flask containing 100 ml autoclaved Luria broth media supplemented with antibiotic (Ampicillin 100 µg/ml) and incubate overnight in a 37°C shaking water bath at 250 rpm.
2. Pour the culture in a 2.0 ml centrifuge tube and centrifuge at 5000 rpm for 20 minutes.
3. Discard the supernatant and wash the pellet in 1.0 ml double distilled water. Centrifuge the solution at 1000 rpm for 5 minutes.
4. Discard the supernatant and resuspend the pellet in 250µl cold alkaline solution I (stored at 4°C). Mix properly so that the pellet dissolves.
5. Add 20 mg of lysozyme in the above solution and mix well. Allow it to incubate on ice for 15 minutes.
6. Add 500 µl of alkaline solution II and mix properly. Mix the solution by flicking.
7. Now add 250 µl of alkaline solution III and incubate it on ice for 15 minutes.
8. Centrifuge the sample at 5000 rpm for 10 minutes and filter the supernatant into an autoclaved 2.0 ml centrifuge tube.
9. Fill the tube with the same amount of ice-cold isopropyl alcohol (stored at-20°C) and incubate in cold (-20°C) for 30 minutes.
10. Centrifuge the sample at 5000 rpm for 15 minutes. Discard the supernatant and air dry the pellet.
11. Resuspend the pellet in 500 µl TE buffer.

Purification of Plasmid DNA using Cesium Chloride

1. When the pellet is suspended completely, measure the mass of plasmid DNA by using a balance. For every gram of plasmid DNA solution, add 1.0 gm of solid cesium chloride. Warm the solution at 30°C to dissolve the CsCl salt.
2. Add 5 µl of 10mg/ml ethidium bromide to the above plasmid DNA solution.
3. Centrifuge the sample at 50,000 rpm for 10-15 hours at 20°C in an ultracentrifuge.
4. After the first run is complete remove the tube carefully and place it in the clamp and insert a needle just below the band. Remove the cap on the tube and pull out the band with the help of needle and syringe.
5. Place the sample in another ultra centrifuge tube and fill the tube with a solution of cesium chloride and TE. Spin at 50,000 rpm for 5 hours.
6. When the run is complete follow the above instructions for removing the bands.
7. Place the bands in a centrifuge tube and add equal volumes of TE and mix well. Now add isoamyl alcohol twice the volume of sample in the tube. Shake well and allow the phases to separate.
8. Pipet off the top pink layer and repeat the extraction with isoamyl alcohol until the top layer is clear and transparent.


Dialysis of Plasmid DNA

1. Prepare pieces of dialysis tubing by rinsing very well in autoclaved distilled water. Tie one end of the tube and carefully suspend he extracted plasmid DNA band into the tubing. Tie the other end too and place the sample in dialysis buffer.
2. Dialyze for at least 6 hours at 4°C using a magnetic stirrer. Repeat the process in new buffer solution.
3. Once the dialysis is complete, remove the liquid from the tubing and transfer it into another tube.
4. Add 1/10 volume of 5 M NaCl and twice the volume of pure ethanol.
5. Centrifuge the samples for 20 minutes at 5000 rpm at 4°C.
6. Discard the supernatant, leaving the pellet.
7. Rinse with 95% Ethanol and resuspend the pellet in 100 μl of TE buffer (pH 8.0) or autoclaved distilled water.

GENE IN INTERSEX

Previous genetic studies indicated intersex (ix) functions only in females and that it acts near the end of the sex determination hierarchy to control somatic sexual
differentiation in Drosophila melanogaster. We have cloned ix and characterized its function genetically, molecularly and biochemically. The ix pre-mRNA is not spliced, and ix mRNA is produced in both sexes. The ix gene encodes a 188 amino acid protein, which has a sequence similar to mammalian proteins thought to function as transcriptional activators, and a Caenorhabditis elegans protein that is thought to function as a transcription factor.

Bringing together the facts that (1) the ix phenotype is femalespecific and (2) functions at the end of the sex determination hierarchy, yet (3) is expressed sex nonspecifically and appears likely to encode a transcription factor with no known DNA-binding domain, leads to the inference that ix may require the female-specific protein product of the doublesex (dsx) gene in order to function. Consistent with this inference, we find that for all sexually
dimorphic cuticular structures examined, ix and dsx are dependent on each other to promote female differentiation. This dependent relationship also holds for the only known direct target of dsx, the Yolk protein (Yp) genes. Using yeast 2 -hybrid assay, immunoprecipitation of recombinant tagged IX and DSX proteins from Drosophila S2 cell extracts, and gel shifts with the tagged IX and DSXF proteins, we demonstrate that IX interacts with DSXF, but not DSXM. Taken together, the above findings strongly suggest that IX and DSXF function in a complex, in which
IX acts as a transcriptional co-factor for the DNA-binding DSXF