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.
| Transformation Service | Transformation Service | Min # of Clones | Cost Per Construct |
|---|---|---|---|
| Transformation Service | Initial selection for drug resistance*. Resistant clones will be propagated through multiple transfers in increased drug concentration. | 5 | contact us |
| 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 | contact us |
| 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 | contact us |
| 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 | contact us |
| 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 | contact us |
*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.