Consider a cotransfection assay with expression vectors for two putative transcription factors that you suspect work together to activate transcription, and with a regulatory region of interest attached to a CAT reporter gene. You also test two deletion () mutants of the regulatory region.
(a) How could you confirm that the transcription factors are transcribed and translated in the transfected cells?
(b) Interpret the results of the CAT assay below. The plus and minus symbols indicate that presence or absence of expression vectors for the listed components in the transfection assay.
(c) How would you demonstrate protein-protein interaction between the two transcription factors in vivo and in vitro? Show sample positive results.
(d) Describe protocols to further characterize direct binding of the proteins to DNA in vitro and in vivo. Show sample results.

Question

Consider a cotransfection assay with expression vectors for two putative transcription factors that you suspect work together to activate transcription, and with a regulatory region of interest attached to a CAT reporter gene. You also test two deletion () mutants of the regulatory region. 
(a) How could you confirm that the transcription factors are transcribed and translated in the transfected cells?
(b) Interpret the results of the CAT assay below. The plus and minus symbols indicate that presence or absence of expression vectors for the listed components in the transfection assay.
  (c) How would you demonstrate protein-protein interaction between the two transcription factors in vivo and in vitro? Show sample positive results.
(d) Describe protocols to further characterize direct binding of the proteins to DNA in vitro and in vivo. Show sample results.

Quizplus · Accepted Answer

The Answer of Consider a cotransfection assay with expression vectors for two putative transcription factors that you suspect work together to activate transcription, and with a regulatory region of interest attached to a CAT reporter gene. You also test two deletion () mutants of the regulatory region. 
(a) How could you confirm that the transcription factors are transcribed and translated in the transfected cells?
(b) Interpret the results of the CAT assay below. The plus and minus symbols indicate that presence or absence of expression vectors for the listed components in the transfection assay.
  (c) How would you demonstrate protein-protein interaction between the two transcription factors in vivo and in vitro? Show sample positive results.
(d) Describe protocols to further characterize direct binding of the proteins to DNA in vitro and in vivo. Show sample results.

Consider a Cotransfection Assay with Expression Vectors for Two Putative