	US 12,473,574 B2
	Methods for modifying a genome
Aris N. Economides, Tarrytown, NY (US); Andrew J. Murphy, Croton-on-Hudson, NY (US); Peter Matthew Lengyel, Brooklyn, NY (US); and Peter H. A. Yang, Tarrytown, NY (US)
Assigned to REGENERON PHARMACEUTICALS, INC., Tarrytown, NY (US)
Filed by REGENERON PHARMACEUTICALS, INC., Tarrytown, NY (US)
Filed on Apr. 1, 2022, as Appl. No. 17/712,003.
Application 17/712,003 is a division of application No. 16/459,236, filed on Jul. 1, 2019, granted, now 11,319,557.
Application 16/459,236 is a continuation of application No. 13/940,609, filed on Jul. 12, 2013, granted, now 10,392,633, issued on Aug. 27, 2019.
Application 13/940,609 is a continuation of application No. 12/915,447, filed on Oct. 29, 2010, abandoned.
Claims priority of provisional application 61/256,078, filed on Oct. 29, 2009.
Prior Publication US 2022/0220510 A1, Jul. 14, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/90 (2006.01); C12N 9/00 (2006.01); C12N 15/63 (2006.01)

CPC C12N 15/907 (2013.01) [C12N 9/00 (2013.01); C12N 15/63 (2013.01); A01K 2217/15 (2013.01); A01K 2217/20 (2013.01); C12N 2800/30 (2013.01); C12N 2800/60 (2013.01); C12N 2840/44 (2013.01)]

30 Claims

1. An in vitro method of creating a conditional allele in the genome of a mouse embryonic stem(ES) cell, comprising:

(1) modifying a target gene of the mouse ES cell by introducing a nucleic acid construct into the target gene by homologous recombination, the nucleic acid construct comprising:

(a) targeting arms for directing the nucleic acid construct to the target gene;

(b) an actuating sequence in sense orientation with respect to transcription of the target gene, wherein the actuating sequence comprises a 3′ splice acceptor followed by a reporter in sense orientation, and a drug selection cassette (DSC) in antisense orientation;

(d) recombinable units that recombine upon exposure to a first recombinase to form the conditional allele, wherein the conditional allele that lacks the actuating sequence and the DSC, and contains the NSI in sense orientation and the COIN element in antisense orientation;

wherein the recombinable units comprise two pairs of cognate recombinase recognition sites recognized by the first recombinase, two pairs of cognate recombinase recognition sites recognized by a second recombinase, and a pair of cognate recombinase recognition sites recognized by a third recombinase, wherein the first recombinase, the second recombinase, and the third recombinase are different from each other;

wherein the nucleic acid construct comprises:

(A) five pairs of cognate recombinase recognition sites:

(a) a first pair of cognate recombinase recognition sites, R1/R1′;

(b) a second pair of cognate recombinase recognition sites, R2/R2′;

(d) a fourth pair of cognate recombinase recognition sites, R4/R4′; and

(e) a fifth pair of cognate recombinase recognition sites R5/R5′,

wherein no pair of cognate recombinase recognition sites is identical to any other pair,

wherein R1/R1′ and R3/R3′ are recognized by the first recombinase, wherein R4/R4′ and R5/R5′ are recognized by the second recombinase, and wherein R2/R2′ are recognized by the third recombinase,

wherein the first, second, and third recombinases are not the same, and

wherein the construct comprises from 5′ to 3′ with respect to orientation on a sense strand: R1; the actuating sequence, wherein the actuating sequence comprises the 3′ splice acceptor followed by the reporter in sense orientation; R2; the DSC in antisense direction; R3; R4; the NSI in antisense orientation; R5; R1′ wherein R1′ is oriented with respect to RI to direct an inversion of sequence between R1 and R1′; R3′ wherein R3′ is oriented with respect to R3 to direct an inversion of sequence between R3 and R340 ; the COIN element in antisense orientation; R5′ wherein R5′ is oriented with respect to R5 to direct an excision of sequence between R5 and R5′; R4′ wherein R4′ is oriented with respect to R4 to direct an excision of sequence between R4 and R4′; and R2′ wherein R2′ is oriented with respect to R2 to direct an excision of sequence between R2 and R2′

(B) five pairs of cognate recombinase recognition sites:

(a) a first pair of cognate recombinase recognition sites, R1/R1′;

(b) a second pair of cognate recombinase recognition sites R2/R2′;

(d) a fourth pair of cognate recombinase recognition sites, R4/R4′; and

(e) a fifth pair of cognate recombinase recognition sites, R5/R5′,

wherein no pair of cognate recombinase recognition sites is identical to any other pair,

wherein R4/R4′and R5/R5′ are recognized by the first recombinase, wherein R2/R2′ and R3/R3′ are recognized by the second recombinase, and wherein R1/R1′ are recognized by the third recombinase,

wherein the first, second, and third recombinases are not the same, and

wherein the construct comprises from 5′ to 3′ with respect to orientation on a sense strand: R1; R2; R3; the COIN element in antisense orientation; R4; R5; R3′ wherein R3′ is oriented with respect to R3 to direct an excision of sequence between R3and R3′; the NSI in antisense orientation; R2′ wherein R2′ is oriented with respect to R2 to direct an excision of sequence between R2 and R2′; R4′ wherein R4′ is oriented with respect to R4 to direct an inversion of sequence between R4 and R4′; the DSC in antisense direction; R1′ wherein R1′ is oriented with respect to R1 to direct an excision of sequence between R1 and R1′; the actuating sequence, wherein the actuating sequence comprises the 3′ splice acceptor followed by the reporter in sense orientation; and

R5′ wherein R5′ is oriented with respect to R5 to direct an inversion of sequence between R5 and R5′;

(a) a first pair of cognate recombinase recognition sites, R1/R1′;

(b) a second pair of cognate recombinase recognition sites R2/R2′;

(d) a fourth pair of cognate recombinase recognition sites, R4/R4′; and

(e) a fifth pair of cognate recombinase recognition sites, R5/R5′,

wherein no pair of cognate recombinase recognition sites is identical to any other pair,

wherein R2/R2′ and R3/R3′ are recognized by the first recombinase, wherein R4/R4′and R5/R5′ are recognized by the second recombinase, and wherein R1/R1′ are recognized by the third recombinase,

wherein the first, second, and third recombinases are not the same, and

wherein the construct comprises, from 5′ to 3′, with respect to the direction of transcription: R1; R2; R3; R4; the NSI in antisense orientation;

R5; R2′ wherein R2′ is oriented with respect to R2′ to direct an inversion of sequence between R2and R2′; the DSC; R1′ wherein R1′ is oriented with respect to R1to direct an excision of sequence between R1and R1′; the actuating sequence, wherein the actuating sequence comprises the 3′ splice acceptor followed by the reporter in sense orientation; R3′ wherein R3′ is oriented with respect to R3to direct an inversion of sequence between R3and R3′; the COIN element in antisense orientation; R5′ wherein R5′ is oriented with respect to R5to direct an excision of sequence between R5and R5′; and R4′ wherein R4′ is oriented with respect to R4to direct an excision of sequence between R4and R4′;

(D) five pairs of cognate recombinase recognition sites:

(a) a first pair of cognate recombinase recognition sites, R1/R1′;

(b) a second pair of cognate recombinase recognition sites R2/R2′;

(d) a fourth pair of cognate recombinase recognition sites, R4/R4′; and

(e) a fifth pair of cognate recombinase recognition sites, R5/R5′,

wherein no pair of cognate recombinase recognition sites is identical to any other pair,

wherein the first, second, and third recombinases are not the same, and

wherein the construct comprises, from 5′ to 3′ with respect to the direction of transcription: R1; the actuating sequence, wherein the actuating sequence comprises the 3′ splice acceptor followed by the reporter in sense orientation; R2; the DSC; R3; the NSI in antisense orientation; R4; R5; R1′ wherein R1′ is oriented with respect to R1to direct an inversion of sequence between R1and R1′; R3′ wherein R3′ is oriented with respect to R3to direct an inversion of sequence between R3and R3′; the COIN element in antisense orientation; R5′ wherein R5′ is oriented with respect to R5to direct an excision of sequence between R5and R5′; R4′ wherein R4′ is oriented with respect to R4to direct an excision of sequence between R4and R4′; and R2′ wherein R2′ is oriented with respect to R2to direct an excision of sequence between R2and R2′;

(E) five pairs of cognate recombinase recognition sites:

(a) a first pair of cognate recombinase recognition sites, R1/R1′;

(b) a second pair of cognate recombinase recognition sites R2/R2′;

(d) a fourth pair of cognate recombinase recognition sites, R4/R4′; and

(e) a fifth pair of cognate recombinase recognition sites, R5/R5′,

wherein no pair of cognate recombinase recognition sites is identical to any other pair,

wherein R4/R4′ and R5/R5′ are recognized by the first recombinase, wherein R2/R2′ and R3/R3′ are recognized by the second recombinase, and wherein R1/R1′ are recognized by the third recombinase,

wherein the first, second, and third recombinases are not the same, and

wherein the construct comprises from 5′ to 3′ with respect to the direction of transcription: R1; R2; R3; the COIN element in antisense orientation; R4; R5; R3′ wherein R3′ is oriented with respect to R3to direct an excision of sequence between R3and R3′; R2′ wherein R2′ is oriented with respect to R2to direct an excision of sequence between R2and R2′; the NSI in antisense orientation; R4′ wherein R4′ is oriented with respect to R4to direct an inversion of sequence between R4and R4′;

the DSC; R1′ wherein R1′ is oriented with respect to R1to direct an excision of sequence between R1and R1′; the actuating sequence, wherein the actuating sequence comprises the 3′ splice acceptor followed by the reporter in sense orientation; and R5′ wherein R5′ is oriented with respect to R5to direct an inversion of sequence between R5and R5′; and or

(F) five pairs of cognate recombinase recognition sites:

(a) a first pair of cognate recombinase recognition sites, R1/R1′;

(b) a second pair of cognate recombinase recognition sites R2/R2′;

(d) a fourth pair of cognate recombinase recognition sites, R4/R4′; and

(e) a fifth pair of cognate recombinase recognition sites, R5/R5′,

wherein R2/R2′ and R3/R3′ are recognized by the first recombinase, wherein R4/R4′ and R5/R5′ are recognized by the second recombinase, and wherein R1/R1′ are recognized by the third recombinase,

wherein no pair of cognate recombinase recognition sites is identical to any other pair,

wherein the first, second, and third recombinases are not the same, and

wherein the construct comprises from 5′ to 3′ with respect to the direction of transcription: R1; R2; R3; the NSI in antisense orientation; R4;

R5; R2′ wherein R2′ is oriented with respect to R2to direct an inversion of sequence between R2and R2′; the DSC; R1′ wherein R1′ is oriented with respect to R1to direct an excision of sequence between R1 and R1′; the actuating sequence, wherein the actuating sequence comprises the 3′ splice acceptor followed by the reporter in sense orientation; R3′ wherein R3′ is oriented with respect to R3to direct an inversion of sequence between R3and R3′; the COIN element in antisense orientation; R5′ wherein R5′ is oriented with respect to R5to direct an excision of sequence between R5 and R5′; and R4′ wherein R4′ is oriented with respect to R4to direct an excision of sequence between R4 and R4′; and

(2) exposing the mouse ES cell to the first recombinase, wherein the recombinable units recombine to form the conditional allele, wherein the conditional allele lacks the actuating sequence and the DSC, and contains the NSI in sense orientation and the COIN element in antisense orientation.