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Efficient Transfection of Neurospora Crassa

Katherine A. Borkovich PhD and Kimberly Vollmer
Katherine A. Borkovich PhD, University of Texas-Houston Medical Center,
Dept. of Microbiology and Molecular Genetics, Houston, Texas.
Kimberly Vollmer, Brinkmann™ Instruments Inc., BioSystems Application Lab, Westbury, New York. INTRODUCTION:

The diverse reproductive capabilities and the nutritional conventions of Neurospora crassa have made this filamentous fungus a model organism in the field of molecular biology and genetics for more than a half a century. In fact, N. crassa was used in the Nobel Prize-winning "One-gene One-enzyme" theory [1]. Since then, N. crassa has been used as a model organism in other types of molecular and genetic applications, joining the illustrious company of E. coli and Drosphila melanogaster.

Perhaps one of the most important roles forN. crassa has been as a host organism for exogenous DNA. Over the years, several techniques have been developed to introduce exogenous material into cells. Electroporation, a technique utilizing electronically regulated pulses to open the cellular membrane and introduce material, has become an easy and efficient method of transfection.

The Eppendorf® Electroporator 2510 employs electric pulse technology to provide an easy and efficient means of introducing exogenous DNA into N. crassa conidia [2]. Most procedures in the past have either used protoplasts for the transfection of N. crassa, or have required the addition of an enzyme to facilitate the weakening of the cell wall. When using the Electroporator 2510, germinated conidia can be transfected efficiently without having to weaken the cell wall or create protoplasts.

OBJECTIVE:

To provide a simple, efficient method for transfecting N. crassa conidia with the Eppendorf Electroporator 2510.

EXPERIMENTAL METHOD:

Cell Cultivation and Harvesting
A Neurospora crassa wild type strain was cultured in VM agar with appropriate supplements in a 250 ml Erlenmeyer flask (a 125 ml flask may also be used) [3]. The flask was incubated at 30°C for 3 days, then transferred to room temperature and exposed to fluorescent light for an additional 5 days. It is necessary to use conidia that are at least eight days old.

The conidia were harvested using sterile water. The suspension was transferred to a 50 ml conical tube and centrifuged at 2,500 rpm for 5 minutes to pellet the conidia. After aspirating the supernatant, the pellet was re-suspended in 25 ml of sterile water. The tube was vortexed to bring the conidia pellet into solution and then re-centrifuged at 2,500 rpm for 5 minutes. The conidia pellet was re-suspended in 25 ml of cold 1 M sorbitol (vortexed for re-suspension) and then centrifuged as stated above; this was repeated twice.

The final pellet was re-suspended in 0.5 ml cold sorbitol and transferred to a 2 ml Eppendorf microcentrifuge tube. The concentration of the suspension was determined using a hemacytometer. The optimal concentration is approximately 2.5 x 109/ml. It may be necessary to concentrate or dilute the sample to bring it to optimal concentration.

Electroporation Procedure
The suspension was transferred in 40 µl aliquots to two Eppendorf 2 ml tubes on ice. In Tube #1, 1 µg of supercoiled DNA (plasmid pCSN44) was added. The selection marker on this plasmid was for hygromycin B resistance [4]. In Tube #2, an equal volume of TE (10 mM TrisCl, 1 mM EDTA, pH 8) was added for control purposes. Both tubes were left on ice, while chilling two 2 mm Eppendorf electroporation cuvettes. The Eppendorf 2510 was set to 2,000 V and the solution from Tube #2 was transferred to a dry, chilled cuvette and then placed in the 2510 (electroporation takes place by pressing the pulse button twice). The cuvette was quickly removed after electroporation and placed on ice. 960 µl of cold sorbitol was added to the cuvette and mixed gently using a Pasteur pipette, then removed and placed in an Eppendorf 2 ml tube. Solution from Tube #1 was then added to a dry, chilled 2 mm cuvette and placed in the 2510 for electroporation. The subsequent steps performed with the sample were identical with the control.

Selection
100 µl of each sample were plated on selective FIGS plates using 10 ml of regeneration agar [5]. The plates were incubated at 30°C for 2 days. Note that these plates must be incubated for at least two days. The number of transfectants was then determined.

RESULTS:

Strain Type Plasmid Marker Number of Transformants/g DNA
Wild Type
(74-OR23-1A)
pCSN44 HPH 370
Wild Type
(74-OR23-1A)
Negative Control N/A 0
DISCUSSION:

Neurospora crassa has been a model organism in genetic studies for many years. It is an organism that shows great promise for its use in many areas of research including pharmaceuticals, food, molecular biology, genetics, and biochemistry. The emergence of an easier method of transferring exogenous DNA into this organism can only facilitate its use in these areas of study.

Electroporation with the Eppendorf 2510 Electroporator offers a rapid, easy method for transfecting N. crassa conidia. It is no longer necessary to create protoplasts, which are fragile after transfection and may not survive standard electroporation procedures. The method also eliminates the need to treat the conidia with enzymes that weaken the cell wall, which may be required in other electroporation protocols. This procedure is a practical alternative that eliminates the use of toxic chemicals and unnecessary steps.

The transfection results recorded show that this procedure is quick, easy, and efficient. It is also important to note that the steps listed in this protocol make it easily adaptable for use with various selection markers, plasmids, and other types of fungi, with small modifications and adjustments.

REFERENCES :

[1] Beadle, G.W., and E.L. Tatum. 1941. Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. USA, 27: 499-506.

[2] Vann, D.C. 1995. Electroporation based transformation of freshly harvested conidia of Neurospora crassa. Fungal Genetics Newsleter. 42A, 53.

[3] Davis, R.H., and F.J. deSerres. 1970. Genetic and microbiological research techniques for Neurospora crassa. Meth. Enzymology, 17:79-143.

[4] Staben C., B. Jensen, M. Singer, J. Pollock, M. Scechtman, J. Kinsey, and E. Selker. 1989. Use of bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation. Fungal Genetics Newsletter. 36: 79-81.

[5] Case, M.E., M.Schweizer, S.R. Rushner, and N.H. Giles. 1979. Efficient transformation of Neurospora crassa by utilizing hybrid DNA. Proc. Nat. Acad. Sci. USA, 76: 5259-5263.


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