Compositions and methods for monitoring in real-time construction and bioengineering of mammalian synthetic chromosomes

Abstract

The present invention encompasses compositions and methods to allow one to monitor formation of synthetic chromosomes in real-time via standardized fluorescent technology, eliminating the need for cumbersome, expensive, and possibly mutagenic analysis.

We claim: 
     
         1 . A mammalian synthetic chromosome production host cell line for tracking synthetic chromosome production in real-time, comprising:
 (i) an endogenous chromosome tag that labels one or more endogenous chromosomes in the chromosome production host cell line; and   (ii) a differently-labeled synthetic chromosome tag that identifies, apart from the endogenous chromosomes, a synthetic chromosome as it is being produced,   wherein the endogenous chromosome tag and synthetic chromosome tag are stably integrated into the synthetic chromosome production host cell line's genome.   
     
     
         2 . The host cell line of  claim 1 , wherein an arm of an endogenous chromosome comprises a recombination site compatible for interaction with a recombination site in the synthetic chromosome. 
     
     
         3 . The host cell line of  claim 1 , comprising a fully-functional mammalian synthetic chromosome. 
     
     
         4 . The host cell line comprising the synthetic chromosome of  claim 3 , wherein the synthetic chromosome-specific tag is stably integrated into the synthetic chromosome. 
     
     
         5 . The host cell line comprising the synthetic chromosome of  claim 3 , wherein both the endogenous chromosome tag and the synthetic chromosome-specific tag are stably integrated into the synthetic chromosome. 
     
     
         6 . The host cell line comprising the synthetic chromosome of  claim 3 , wherein the endogenous chromosome tag has been stably integrated into the synthetic chromosome and the synthetic chromosome-specific tag has been stably integrated into the synthetic chromosome production host cell line's genome. 
     
     
         7 . The host cell line of  claim 1 , wherein the endogenous chromosome tag and the synthetic chromosome-specific tag comprise fluorescent tags, and the monitoring step is performed by fluorescence microscopy. 
     
     
         8 . The host cell line of  claim 7 , wherein the fluorescent tags are selected from TagBFP, TagCFP, TagGFP2, TagYFP, TagRFP, FusionRed, mKate2, TurboGFP, TurboYFP, TurboRFP, TurboFP602, TurboFP635, TurboFP650, AmCyan1, AcvGFP1, ZsGreen1, ZsYellow1, mBanana, mOrange, mOrange2, DsRed-Express2, DsRed-Express, tdTomato, DsRed-Monomer, DsRed2, AsRed2, mStrawberry, mCherry, HcRed1, mRaspberry, E2-Crimson, mPlum, Dendra 2, Timer, PAmCherry, HALO-tags, infrared-shifted fluorescent proteins, and the like. 
     
     
         9 . The host cell line of  claim 1 , wherein the tags of the endogenous chromosome tag and the synthetic chromosome-specific tag comprise:
 (i) chemiluminescent tags and the monitoring step is performed by chemiluminescent microscopy, or   (ii) phosphorescent tags and the monitoring step is performed by phosphorescent microscopy.   
     
     
         10 . The host cell line of  claim 1 , wherein the endogenous chromosome tag comprises a marker specific to histones H1, H2A, H2B, H3, H4, or H5. 
     
     
         11 . The host cell line of  claim 1 , wherein the synthetic chromosome-specific tag comprises 4 n  nucleotides that have been screened against a database of known sequences. 
     
     
         12 . The host cell line of  claim 1 , wherein the cell line is selected from an animal cell line, a plant cell line, a mammalian cell line, a rodent cell line, a zebrafish cell line, an embryonic cell line, a pluripotent cell line, an adult-derived stem cell, a reprogrammed cell line and a human cell line. 
     
     
         13 . The host cell line of  claim 12 , wherein the human cell line is HT1080. 
     
     
         14 . The host cell line of  claim 1 , wherein the cell line produces synthetic chromosomes via an approach selected from: a top down approach, a bottom up approach, engineering of naturally occurring minichromosomes, or de novo chromosome generation by targeted amplification of chromosomal segments. 
     
     
         15 . The host cell line of  claim 14 , wherein the cell line produces synthetic chromosomes via de novo chromosome generation by targeted amplification of pericentric chromosomal segments. 
     
     
         16 . The host cell line of  claim 1 , wherein the endogenous chromosome tag comprises a plurality of endogenous chromosome tags. 
     
     
         17 . The host cell line of  claim 1 , wherein the endogenous chromosome tag is a fusion protein, nucleic acid/protein chimera, nucleic acid/protein complex, a RNA/CRISPR-CAS9 complex, or a moiety comprising a TALEN protein specific to the endogenous chromosomes, and a fluorescent or phosphorescent label. 
     
     
         18 . The host cell line of  claim 1 , further comprising one or more of: an endogenous chromosome binding moiety; a first reporter moiety; a second reporter moiety; a synthetic chromosome binding moiety; and a selectable marker. 
     
     
         19 . A fully-functional mammalian synthetic chromosome, produced by transfection of synthetic chromosome production components into the host cell line of  claim 1 . 
     
     
         20 . The fully-functional mammalian synthetic chromosome of  claim 19 , wherein the synthetic chromosome is 1-120 Mbp in size.