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Transformation Services

The Tetrahymena Stock Center (TSC) is now providing biolistic transformation services to facilitate genetic engineering in this versatile research organism. Functional gene analysis frequently relies on research strategies like gene disruption or modification, gene knock-ins and knock-outs, overexpression of proteins, and gene tagging. Genetic transformation is a critical technology often required in the application of these strategies. TSC is now set up to provide macronuclear and micronuclear transformations as a fee-based service for academic researchers.

Fee-based Services
Minimum Fee
Molecular Analysis
Placing an Order
Transformation vectors
DNA
DNA Preparation
Shipping DNA
Packaging

Transformation Service Selection Options Minimum # of Clones+ Cost per Construct
Macronuclear (Somatic) Transformation
Not tested for complete replacement.
Initial selection for drug resistance*. Resistant clones will be propagated through multiple transfers in increased drug concentration. 5 $500
Macronuclear (Somatic) Modification
Pushed to “complete” replacement as determined by PCR**
Initial selection for resistance to marker of choice*. Resistant clones will be pushed to complete replacement based on PCR analysis (primer sequences supplied by client). 5 $650
Macronuclear transformation using high-copy number rDNA-based vector
rDNA vector pD5H8 is available from TSC
Paromomycin resistance based on mutation in the rDNA vector. Other selection cassettes can be introduced with gene. 5 $500
Micronuclear (Germline) Transformation
Micronuclear transformation is required for germline transmission of introduced constructs.

Heterozygous clones
Initial selection based on dual resistance to selection marker and a micronuclear drug resistance gene. Heritability confirmed by outcross mating to insure meiotic transmission of the construct. 1 $750
Micronuclear (Germline) Transformation
Micronuclear transformation is required for germline transmission of introduced constructs.

Homozygous clones
Initial selection based on dual resistance to selection marker and a micronuclear drug resistance gene. Micronuclear construct made homozygous using a “star” outcross mating. 1 $1000
+A minimum of 5 independent macronuclear or 1 independent micronuclear transgenic clones will be provided for each construct. If more resistant clones are produced (very likely for macronuclear constructs) additional clones can be provided if desired.

*Paromomycin or cycloheximide is recommended for transformant selection.  Puromycin is also an option but there may be an additional charge for the higher priced drug. Plasmids containing constitutive and inducible selection cassettes using these 3 resistance genes are available through TSC. If an alternate selection scheme is necessary, check with TSC regarding the possible use of blasticidin or taxol.

**Macronuclear transformants will be selected in the drug of choice and subsequently grown in at least 2x the minimal drug concentration used for initial selection. Complete replacement will not be attempted unless specifically requested. Because of the increased time and expense associated with producing completely assorted clones, there is an additional charge for this service. Complete replacement is dependent on a number of factors, including the target site and the transforming vector used and may not be possible for some constructs. Although the TSC staff is happy to provide input, the final determination to attempt complete replacement is the responsibility of the client.

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Minimum Fee

The TSC staff is experienced in all aspects of Tetrahymena transformation and will make every effort to successfully produce the desired transformant clones. However, on occasion, unforeseen obstacles can arise that may prevent the creation of the desired transformant. Based on our experience, transformation problems most often arise as a result of incorrectly designed or prepared vector constructs, or attempts to manipulate a gene that results in lethality or poor cell growth. There also appear to be a few rare loci in Tetrahymena that are not easily amenable to transformation, for unknown reasons. Unfortunately, it is often impossible to determine if a transformation will succeed until a significant amount of time and effort have already been invested. In the event that a transformant cannot be obtained, following consultation with the client, the project will be terminated and a fee (25 -50% of the original cost) will be charged depending upon the effort (labor and supplies) invested in the project. (back to top)

Molecular Analysis

Transformant clones are selected based on drug resistance determined by the selection cassette used in the transformation construct. The level of replacement by the transgene construct is not determined unless complete replacement is requested. Complete replacement is monitored by PCR analysis across the insertion site, using primer sequences provided by the client. Note that complete replacement is dependent on a number of factors, including the target site and the transforming vector used, and may not be possible for some constructs. (back to top)

Placing an Order

To request a transformation, please complete the transformation request worksheet. Transformation requests will be performed in the order they are received. We strongly urge those interested in our transformation services to contact us before ordering to discuss details of the proposed project. All information regarding individual projects will be kept confidential. (back to top)

Transformation vectors

Tetrahymena can be transformed by homologous recombination at specifically targeted sites or with autonomously replicating vectors. Some commonly used plasmids that can serve as a starting point for some types of construct creation are available through the Stock Center. Clients are responsible for the design and construction of their transformation vector.  Vector design is critical for successful transformation. Please contact us if you have any questions regarding vector selection, design, or preparation. (back to top)

DNA

A minimum of 30 µg of purified vector at a concentration of ~ 1 µg/µl is required. DNA for transformation must be of high quality and purity. See instructions for the preparation of vector DNA for transformation below. (back to top)

DNA Preparation

  1. Purify plasmid DNA using a commercial plasmid DNA preparation kit. Since free DNA ends catalyze homologous recombination, to optimize transformation efficiency vectors targeted for homologous recombination should be cut with restriction enzymes that release the complete transformation cassette. The enzyme should be removed by phenol/chloroform extraction and ethanol precipitation or other means of purification, and the DNA should be resuspended in ddH2O at ~ 1 µg/µl. It is not necessary to purify the insert away from the vector backbone. Autonomously replicating vectors should be provided as super coiled circular plasmid. Dissolve DNA in ddH2O. High quality, high purity DNA is required for successful biolistic transformation.
  2. Measure DNA concentration using a spectrophotometer. An acceptable DNA purity is within an OD260/OD280 range of 1.7 to 2.0. Dilute DNA to 0.8 - 1 µg/µl concentration.
  3. Check DNA quality by electrophoresis. Include ~ 0.5 -1 µg/lane of the following on your gel:
    1. undigested DNA
    2. DNA digested with 2-3 restriction enzymes to confirm the construct.
    3. DNA ladder
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Shipping DNA

Ship DNA by overnight express (ice is not needed) to:

Tetrahymena Stock Center
C5 152 Veterinary Medical Center
930 Campus Rd.
Cornell University
Ithaca, New York 14853

The package should include the following:

  1. A minimum of 30 µg DNA in ddH2O (concentration of 0.8 – 1 µg/µl), packaged as indicated below.
  2. Construct map showing
    1. important restriction sites
    2. size and organization of the construct, including, if relevant, promoter and 3’ polyA regions
    3. size and location of flanking regions used for targeting
    4. antibiotics for bacterial selection
    5. selectable cassette or marker
  3. OD260/OD280 values and ratio
  4. Gel image illustrating DNA quality (as described in DNA Preparation above).
  5. Brief overview of the project, including vector type, transformation type (micronuclear or macronuclear, KO, gene replacement, gene tagging, exogenous gene expression, etc), target site, and project goals (for example, mutation, partial or complete replacement, gene expression).
  6. Target strain requirements. Contact us if you have specific strain requirements.
  7. Complete contact information including
    1. name of PI, office phone, fax, e-mail (for billing)
    2. name of contact person (if not PI), lab/office phone, e-mail (for construct communications and receiving samples)
    3. complete mailing address, including street numbers, if applicable
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Packaging

Plasmid DNA should be placed in a clearly labeled primary 1.5 ml tube and secured with parafilm. The tube should then be placed in a sealable secondary container (a sealable plastic bag will do) and shipped in a cushioned envelope or small box. Please enclose an itemized list of package contents along with complete sender identification in the package.

You will receive an email when your DNA is received with a project ID number. The project ID number will be used in all subsequent communications regarding your construct. (back to top)