US 11,054,424 B2
Cell identification method
Monsef Benkirane, Saint Gely du Fesc (FR); and Gael Petitjean, Montpellier (FR)
Assigned to CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS), Paris (FR)
Appl. No. 15/538,742
Filed by Centre National De La Recherche Scientifique (CNRS), Paris (FR)
PCT Filed Dec. 17, 2015, PCT No. PCT/FR2015/053579
§ 371(c)(1), (2) Date Jun. 22, 2017,
PCT Pub. No. WO2016/102829, PCT Pub. Date Jun. 30, 2016.
Claims priority of application No. 1463138 (FR), filed on Dec. 22, 2014.
Prior Publication US 2017/0350889 A1, Dec. 7, 2017
Int. Cl. C12Q 1/68 (2018.01); G01N 33/569 (2006.01); C12Q 1/70 (2006.01); C12Q 1/6897 (2018.01); C07K 16/10 (2006.01); C12N 7/04 (2006.01); G01N 33/50 (2006.01)
CPC G01N 33/56988 (2013.01) [C07K 16/1045 (2013.01); C12N 7/04 (2013.01); C12Q 1/6897 (2013.01); C12Q 1/703 (2013.01); G01N 33/505 (2013.01); G01N 33/569 (2013.01); G01N 33/56972 (2013.01); C12Q 2521/507 (2013.01); C12Q 2600/136 (2013.01); G01N 2333/16 (2013.01); G01N 2333/70514 (2013.01); G01N 2333/70517 (2013.01); G01N 2333/70589 (2013.01); G01N 2333/70596 (2013.01); G01N 2500/10 (2013.01)] 6 Claims
 
1. A method for detection of cells that constitute the reservoir of a virus inducing immunodeficiency in a donor mammal by using a recombinant virus expressing site-specific recombinase, the donor mammal being a human, the method comprising:
(a) introducing into hematopoietic stem cells from the donor mammal:
a nucleic acid molecule comprising a first reporter gene, the nucleic acid molecule comprising a first sequence encoding the first reporter gene, under the control of at least one element necessary for transcription, the first sequence being bordered by:
at least one first pair of sequences targeting a site-specific recombinase, the first pair comprising a P1-1 sequence and a P1-2 sequence,
at least one second pair of sequences targeting a site-specific recombinase, the second pair comprising a P2-1 sequence and a P2-2 sequence,
the sequences of each of the first and second pairs of sequences being oppositely oriented relative to one another,
the sequences of the first pair of sequences targeting a site-specific recombinase being unable to recombine with the sequences of the second pair of sequences targeting a site-specific recombinase, and the sequences of the second pair of sequences targeting a site-specific recombinase being unable to recombine with the sequences of the first pair of sequences targeting a site-specific recombinase,
one sequence from the first pair and one sequence from the second pair of sequences targeting a site-specific recombinase being located upstream of the first nucleic acid sequence, and one sequence from the first pair and one sequence from the second pair of sequences targeting a site-specific recombinase being located downstream of the first nucleic acid sequence,
such that the sequences of the same pair never flank the two sequences of the other pair,
the sequence of the first nucleic acid molecule being such that, in the absence of combination induced by the site-specific recombinase, it has an open reading frame coding for the first reporter gene in a 3′-5′ orientation;
(b) myeloablating the hematopoietic system of a recipient mammal, the recipient mammal being a mouse, and reconstructing the hematopoietic system of the recipient mammal, said reconstructing using the hematopoietic stem cells of the donor mammal prepared in (a);
(c) infecting the recipient mammal having a reconstructed bone marrow in (b) with a virus inducing an immunodeficiency in the donor mammal, the virus expressing a site-specific recombinase;
(d) treating the recipient mammal infected in (c) with a treatment inhibiting development of the virus; and
(e) detecting, in the recipient mammal, hematopoietic cells expressing the first reporter and being resistant to the treatment inhibiting development of the virus.