JP2016010330A5

JP2016010330A5 -

Info

Publication number: JP2016010330A5
Application number: JP2014132622A
Authority: JP
Filing date: 2014-06-27
Publication date: 2017-08-10
Anticipated expiration: 2034-06-27

Claims

A method of expressing a plurality of foreign genes in an embryoid body composed of human induced pluripotent stem cells (iPS cells),
1) Transfecting an iPS cell with a foreign gene using a non-liposomal cationic lipid transfection reagent using an episomal vector,
2) a step of selecting iPS cells transfected with a foreign gene using a drug selection marker and / or a reporter gene, and 3) culturing the selected iPS cells by a non-static culture method, A method comprising the step of growing to the body.

2. The method according to claim 1, wherein two or more foreign genes are used, and an episomal vector construct is prepared by combining different drug selection markers and / or reporter genes for each gene.

The foreign gene comprises two kinds of foreign genes, and includes a nucleic acid selected from a drug selection marker different for each foreign gene and / or a reporter gene having the sequence of SEQ ID NO: 7 or 8. 2. The method according to 2.

The method according to any one of claims 1 to 3, wherein the foreign gene is selected from transcription factors that induce differentiation.

The method according to claim 4, wherein the transcription factor is two or more selected from the group consisting of FOXA2, GATA4, HEX and C / EBPα.

The method according to any one of claims 1 to 5, wherein the non-static culture method is a rotary culture method.

The method according to claim 6, wherein a rotational speed at the time of culture in the rotary culture method is 2.5 to 9.9 rpm.

The iPS cell culture is performed by adding a suspension of iPS cells having a cell concentration of 1.6 to 160 × 10 ³ cells / mL, and culturing the iPS cells. The method described in 1.

An embryoid body into which a foreign gene consisting of human induced pluripotent stem cells (iPS cells) has been introduced,
1) Transfecting an iPS cell with a foreign gene using a non-liposomal cationic lipid transfection reagent using an episomal vector,
2) selecting iPS cells transfected with a foreign gene using a drug selection marker and / or a reporter gene; and
3) A step of culturing selected iPS cells by a non-static culture method and allowing them to grow to embryoid bodies
An embryoid body produced by a method comprising:

A method of expressing a plurality of foreign genes in an embryoid body composed of human induced pluripotent stem cells (iPS cells),
1) In episomal vector,
(i) foreign genes,
(ii) a drug selection marker for G418® or hygromycin B, and
(iii) Multiple types of reporter genes (i) to (iii) incorporated in combination with a nucleic acid sequence encoding EGFP (SEQ ID NO: 7) or a nucleic acid sequence encoding Cherry Picker (registered trademark) (SEQ ID NO: 8) Producing an episomal vector construct of
2) transfecting a plurality of foreign genes into iPS cells by transfecting a plurality of episomal vector constructs into iPS cells using a non-liposomal cationic lipid transfection reagent;
3) Corresponding to the combination of (i) to (iii),
Using drug selection markers by the addition of G418 and / or hygromycin B, and / or
Using a reporter gene with green and / or red fluorescence as an indicator,
Selecting iPS cells into which the plurality of foreign genes have been introduced, and
4) A step of culturing the selected iPS cells at a cell concentration of 1.6 to 160 × 10 ³ cells / mL by a rotary culture method at a rotational speed of 2.5 to 9.9 rpm, and proliferating to an embryoid body. Including methods.

An embryoid body into which a foreign gene consisting of human induced pluripotent stem cells (iPS cells) has been introduced,
1) In episomal vector,
(i) foreign genes,
(ii) a drug selection marker for G418® or hygromycin B, and
(iii) Multiple types of reporter genes (i) to (iii) incorporated in combination with a nucleic acid sequence encoding EGFP (SEQ ID NO: 7) or a nucleic acid sequence encoding Cherry Picker (registered trademark) (SEQ ID NO: 8) Producing an episomal vector construct of
2) transfecting a plurality of foreign genes into iPS cells by transfecting a plurality of episomal vector constructs into iPS cells using a non-liposomal cationic lipid transfection reagent;
3) Corresponding to the combination of (i) to (iii),
Using drug selection markers by the addition of G418 and / or hygromycin B, and / or
Using a reporter gene with green and / or red fluorescence as an indicator,
Selecting iPS cells into which the plurality of foreign genes have been introduced, and
4) Select iPS cells from 1.6 to 160 × 10 6 ^３3 A step of cultivating the cells to the embryoid body by culturing by a rotational culture method at a cell rotation rate of 2.5 to 9.9 rpm at a cell concentration of 1 cell / mL
An embryoid body produced by a method comprising: