Microfluidic analysis of ligand induced cell expression

Patent metadata

Field	Value
Publication number	US-12083512-B2
Application number	US-201716069824-A
Country	US
Kind code	B2
Filing date	Jan 13, 2017
Priority date	Jan 14, 2016
Publication date	Sep 10, 2024
Grant date	Sep 10, 2024

Abstract

The present invention relates to the field of microfluidics and in particular to analysing the gene expression of a cell in response to a ligand expressed in the same microfluidic compartment. By barcoding the transcriptome of the cell and of the expression system generating the ligand, the effect of the ligand on the cell expression can be discerned. The invention provides microfluidic compartments and methods for this purpose.

First claim

The invention claimed is:

1. A plurality of microfluidic compartments, wherein at least 1% of said compartments form a subset in which each compartment is a droplet comprising:
(i) a first cell,
(ii) one second cell, the second cell expressing a polypeptide ligand intended to specifically bind to a molecule or part thereof accessible on a surface of the first cell, wherein the one second cell is a cell of B-cell lineage and
(iii) a set of barcode oligonucleotides each comprising a barcode sequence unique to the set and a sequence capable of binding specifically to mRNA and/or cDNA of the first cell and mRNA and/or cDNA of the one second cell.

2. The plurality of microfluidic compartments of claim 1 , wherein the subset is at least 5% of the plurality of microfluidic compartments.

3. The plurality of microfluidic compartments of claim 1 , wherein the first cell and the second cell are derived from different species.

4. The plurality of microfluidic compartments of claim 1 , wherein the molecule or part thereof accessible on the surface of the first cell is a cell signaling receptor.

5. The plurality of microfluidic compartments of claim 1 , wherein the first cell is a diseased cell, a stem or pluripotent cell, or a cell in which pluripotency is inducible by a polypeptide ligand.

6. The plurality of microfluidic compartments of claim 1 , wherein the second cell is a non-human plasma cell of the B-cell lineage and the polypeptide ligand is an antibody produced by the plasma cell.

7. The plurality of microfluidic compartments of claim 1 , wherein the polypeptide ligand is selected from the group consisting of an antibody, an antibody derivative and an antibody mimetic.

8. The plurality of microfluidic compartments of claim 1 , wherein the set of barcode oligonucleotides is linked to a bead.

9. The plurality of microfluidic compartments of claim 1 , wherein said sequence capable of binding specifically to mRNA and/or cDNA is a sequence capable of binding specifically to an mRNA 3′ poly(A) tail or to a gene-specific sequence.

10. The plurality of microfluidic compartments of claim 1 , the compartments of said subset of the plurality of compartments each further comprising one or more of the following:
a cell lytic agent,
an RNase-inhibitor,
a DNase,
reagents for a reverse transcription reaction, and/or
a drug or drug candidate for treating the diseased cell or the disease the diseased cell is derived from, wherein the diseased cell it the first cell.

11. A method for generating a plurality of microfluidic compartments according to claim 1 , comprising the steps of:
(a) introducing into a microfluidic system: (i) a fluid comprising a plurality of first cells, (ii) a fluid comprising a plurality of second cells each expressing a polypeptide ligand which is intended to specifically bind to a molecule or part thereof accessible on the surface of a first cell, and (iii) a fluid comprising sets of barcode oligonucleotides, wherein the barcode oligonucleotides of each set comprises a barcode sequence unique to the set, and a sequence capable of binding specifically to mRNA and/or cDNA of the first cell and mRNA and/or cDNA of the one second cell, and
(b) repeatedly co-compartmentalizing one of the first cells, one of the second cells, and a set of the barcode oligonucleotides into microfluidic compartments, such that the size of the subset of compartments in the plurality of compartments is at least 1%.

12. A method for determining the gene expression of a cell, comprising the steps:
(a) providing a plurality of microfluidic compartments wherein at least 1% of said compartments form a subset in which each compartment is a droplet comprising:
(i) a first cell,
(ii) one second cell or one cell-free expression system, the second cell or the cell-free expression system expressing a polypeptide ligand intended to specifically bind to a molecule or part thereof accessible on a surface of the first cell, and
(iii) a set of barcode oligonucleotides each comprising a barcode sequence unique to the set and a sequence capable of binding specifically to mRNA and/or cDNA of the first cell and mRNA and/or cDNA of the one second cell or the one cell-free expression system,

(b) lysing the cells comprised in the compartments,
(c) reverse transcribing mRNA released from the cells to cDNA,
(d) amplifying the cDNA,
(e) determining the sequence and optionally the respective amount of the cDNA, and
(f) selecting sequences comprising the same barcode sequence, wherein the following sequences are excluded:
sequences derived from a second cell or a cell-free expression system,
sequences comprising a unique barcode sequence that is not associated in other sequences with a nucleotide sequence encoding for a polypeptide ligand expressed by a second cell or a cell-free expression system,
sequences comprising a unique barcode sequence that is associated with a nucleotide sequence encoding for more than one polypeptide ligand expressed by a second cell or a cell-free expression system,

wherein step (c) or step (d) is carried out with a barcode oligonucleotide as a primer.

13. The method of claim 12 , wherein the barcode oligonucleotide has a poly(dT) or poly(dU) sequence if it is used as a primer in step (c) or wherein the barcode oligonucleotide is part of a gene-specific primer pair if it is used as a primer in step (d).

14. The method of claim 12 , wherein the contents of the compartments are pooled after annealing of the barcode oligonucleotide primer and at any stage prior to step (e).

15. The method of claim 12 wherein the method is for determining at least one of: (i) the effect of antibodies on the gene expression of a target cell, wherein the antibodies were raised by immunizing a vertebrate with said target cell or one or more molecules or parts therefrom accessible on its surface, or (ii) the effect of polypeptide ligands on the gene expression of a target cell, wherein the polypeptide ligand is derived from a library.

16. The plurality of microfluidic compartments of claim 1 , wherein the sequence capable of binding specifically to mRNA and/or cDNA is at a 3′ end of the barcode oligonucleotide, and the sequence capable of binding specifically to mRNA and/or cDNA is capable of priming a DNA polymerization.

17. A plurality of microfluidic compartments, wherein at least 1% of said compartments form a subset in which each compartment is a droplet comprising:
(i) a first cell,
(ii) one second cell or one cell-free expression system, the second cell or the cell-free expression system expressing a polypeptide ligand intended to specifically bind to a molecule or part thereof accessible on a surface of the first cell, and
(iii) a set of barcode oligonucleotides each comprising a barcode sequence unique to the set and a sequence capable of binding specifically to mRNA and/or cDNA of the first cell and/or of the one second cell or the one cell-free expression system,
wherein the compartment volume of the subset and the cell concentration of the first cell (i), the cell concentration or cell-free expression system concentration of the one second cell or the one cell-free expression system (ii), and the set concentration of the set of barcode oligonucleotides (iii) is such that λ is between 0.1 and 2 for each of the cell concentration of the first cell (i), the cell concentration of the one second cell or cell-free expression system concentration of the one cell-free expression system (ii), and the set concentration of the set of barcode oligonucleotides (iii),
wherein λ is the average num

Assignees

• European Molecular Biology Laboratory

Inventors

• Hu Hongxing
• Seah Samantha
• Merten Christoph A

Classifications

• C40B20/08

  Direct analysis of the library members per se by physical methods, e.g. spectroscopy · CPC title

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title