Navigation Links
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.


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


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.

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.


Strain Type Plasmid Marker Number of Transformants/g DNA
Wild Type
pCSN44 HPH 370
Wild Type
Negative Control N/A 0

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.


[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.



Page: All 1 2 3 4 5

Related biology technology :

1. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
2. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
3. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
4. Efficient Transfection of Neurospora Crassa
5. Efficient Recovery of Ultrapure Plasmid DNA
6. Efficient and Reliable PCR Setup Using Eppendorf MasterMix
7. Efficient DNA transfection of primary CNS neurons using TransMessenger Transfection Reagent
8. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
9. Cloning Based on Efficient Three-Fragment Assembly DNA Ligation
10. Efficient and Reliable Linear Amplification of cRNA
11. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
Post Your Comments:

(Date:11/27/2015)... , November 27, 2015 ... Growing popularity of companion diagnostics is ... cancer biomarkers market with pharmaceutical companies and ... companion diagnostic tests. . ... Complete report on global cancer biomarkers ...
(Date:11/26/2015)... November 26, 2015 --> ... specializing in imaging technologies, announced today that it has received ... of the Horizon 2020 European Union Framework Programme for Research ... clinical trial in breast cancer. , --> ... --> --> The study aims ...
(Date:11/25/2015)... Nov. 25, 2015  PharmAthene, Inc. (NYSE MKT: PIP) ... a stockholder rights plan (Rights Plan) in an effort ... carryforwards (NOLs) under Section 382 of the Internal Revenue ... PharmAthene,s use of its NOLs could be substantially ... defined in Section 382 of the Code. In general, ...
(Date:11/25/2015)... , November 25, 2015 Studies ... and human plaque and pave the way for more effective ... in cats     --> ... commonly diagnosed health problems in cats, yet relatively little was ... Two collaborative studies have been conducted by researchers from the ...
Breaking Biology Technology:
(Date:11/12/2015)...  Arxspan has entered into an agreement with ... use of its ArxLab cloud-based suite of biological ... will support the institute,s efforts to electronically manage ... internally and with external collaborators. The ArxLab suite ... Institute,s electronic laboratory notebook, compound and assay registration, ...
(Date:11/10/2015)... Nov. 10, 2015 About ... that helps to identify and verify the identity ... considered as the secure and accurate method of ... a particular individual because each individual,s signature is ... especially when dynamic signature of an individual is ...
(Date:11/2/2015)... Nov. 2, 2015  SRI International has been awarded ... preclinical development services to the National Cancer Institute (NCI) ... provide scientific expertise, modern testing and support facilities, and ... pharmacology and toxicology studies to evaluate potential cancer prevention ... The PREVENT Cancer Drug Development Program is an NCI-supported ...
Breaking Biology News(10 mins):