US 11,702,666 B2
Cell-based assay for determining activity in the retinoblastoma pathway
Tatyana Naryshkina, East Brunswick, NJ (US)
Assigned to Medical Diagnostic Laboratories, LLC, Hamilton, NJ (US)
Filed by Medical Diagnostic Laboratories, LLC, Hamilton, NJ (US)
Filed on Oct. 15, 2021, as Appl. No. 17/502,217.
Application 17/502,217 is a continuation of application No. 16/192,993, filed on Nov. 16, 2018, granted, now 11,180,766.
Claims priority of provisional application 62/756,750, filed on Nov. 7, 2018.
Claims priority of provisional application 62/587,107, filed on Nov. 16, 2017.
Prior Publication US 2022/0073935 A1, Mar. 10, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/79 (2006.01); C12N 9/02 (2006.01); G01N 33/50 (2006.01); C12Q 1/48 (2006.01); C12N 9/12 (2006.01)
CPC C12N 15/79 (2013.01) [C12N 9/0069 (2013.01); C12N 9/12 (2013.01); C12Q 1/485 (2013.01); G01N 33/5005 (2013.01); C07K 2319/60 (2013.01); C12Y 113/12009 (2013.01)] 3 Claims
OG exemplary drawing
 
1. A method of determining the activity of a Rb variant in a cell, comprising the steps of:
a. providing a mammalian cell, said cell comprising a stably integrated first construct, wherein said first construct comprises a first cDNA encoding E2F1 protein which is linked to a first portion of a luciferase gene, and a second cDNA encoding Dp-1 protein;
b. preparing a linear third cDNA of a Rb variant, wherein said Rb variant contains at least one mutation as compared to wild-type Rb, and said third cDNA is linked to a second portion of a luciferase gene to form a second construct, wherein when said first construct is in close proximity with said second construct a complex is formed that emits light;
c. transfecting said second construct into said mammalian cell; and
d. determining the activity of said Rb variant by measuring any generated light emission, wherein an increase in light emission relative to a control mammalian cell is indicative of said Rb variant activity.