ʻOihana Kūkulu Laina Kelepona Paʻa

Ua komo nui ʻo Alpha Lifetech i ke kahua o ka hoʻomohala ʻana i ke kaʻina hana kūkulu laina kelepona paʻa no nā makahiki he nui. Ua kūkulu ʻo ia i kahi kahua hana kūkulu laina kelepona paʻa kūpono, me nā ʻano cell tumor a me nā cell mua ma mua o 300, hiki ke hāʻawi i nā laina cell paʻa me ka mālama ʻana i ka manawa no ka noiʻi ʻepekema a i ʻole ka noiʻi papahana, a me ka hoʻomaʻamaʻa ʻana i ka hana ma hope. Hiki iā Alpha Lifetech ke hāʻawi i nā mea kūʻai aku me nā lawelawe e like me ka hoʻomohala ʻana i ke kaʻina hana strain, ka nānā ʻana i ke strain, ke kūkulu ʻana i ke strain, ka protein recombinant, a me ka hana antibody, e hāʻawi ana i kahi kumu noiʻi no nā lāʻau lapaʻau hou.

Loaʻa iā Alpha Lifetech kahi kahua cell i loaʻa i ka lentivirus, hiki ke hoʻopilikino i ke kaʻina hana hoʻokolohua no nā ʻano like ʻole o nā ʻano transmutation cell stable. Ua hoʻohana mākou i ka ʻenehana knockout gene e kūkulu i nā laina cell knockout CRISPR/Cas9. Ua hoʻohana ʻia nā ʻano hana gene overexpression a me gene silencing e hoʻopaʻa i nā ʻano transmutation. Hoʻopau ka hoʻohui ʻana o ka pūnaewele gene i nā lawelawe kūkulu laina cell vertex integration.

Hoʻolauna i ke kūkulu ʻana i ka laina cell paʻa

ʻO ka laina cell paʻa e pili ana i ka laina cell e hoʻohui i nā genes haole i loko o ka genome cell host i hiki ke hōʻike paʻa ʻia nā genes haole i loko o ke cell host no ka manawa lōʻihi. ʻO ke kumumanaʻo, ʻo ia ka hoʻemi ʻana i ka gene haole i kahi vector me kahi kūʻē, transfect cell host, hoʻohui i ka gene haole i loko o ka chromosome host, a nānā i nā genes kūʻē i loko o ka vector e loaʻa ai nā laina cell paʻa me ka hōʻike kiʻekiʻe o ka protein target a i ʻole ka hoʻopau paʻa ʻana o ka gene target e hoʻokō i nā koi o ka hoʻokolohua.

Loaʻa iā Alpha Lifetech kahi hale hana mālama kūʻokoʻa, ka ʻōnaehana hoʻopaʻa a me ka mālama ʻana o nā cell, a ʻoi aku ma mua o 300 mau ʻano o nā cell tumor a me nā cell mua no ʻoe e koho ai, i hoʻolaʻa ʻia no kāu noiʻi ʻepekema.

Ke kaʻina hana kūkulu laina kelepona paʻa

Ke ʻano o ke kūkulu ʻana i ka laina cell paʻa

ʻO nā ʻano hana kūkulu laina cell paʻa e komo pū me ka lentivirus-mediated, transposon-mediated, CRISPR/Cas9-mediated, a me ka plasmid screening.

ʻO ka lentivirus-mediated kekahi o nā ʻano hana i hoʻohana pinepine ʻia e kūkulu i nā laina cell paʻa. Loaʻa i kēia ʻano hana ka pono o ka hoʻopili ʻana i ka aneane o nā ʻano cell āpau a me ka hoʻohui ʻana i nā mea genetic i loko o ka genome cell host no ka hōʻike paʻa ʻana i kahi manawa lōʻihi. Eia nō naʻe, he mau palena ko kēia ʻōnaehana, a he palena ko ka hiki ke hoʻopili ʻana o lentivirus, no laila ʻaʻole kūpono ia no ka transcription o nā genes me nā wahi transcription lōʻihi

Ke hoʻohālikelike ʻia me ka lentivirus-mediated, ʻaʻohe palena o ka nui o ka gene i hoʻopili ʻia e ka transposon, a me ka hoʻomau ʻana o ka hoʻonui ʻana o ka ʻenehana, ua hoʻomaikaʻi ʻia ka pono o ka hoʻokomo ʻana o ka transposon i nā manawa he nui. ʻO ka hemahema, he paʻakikī ke transfect i kekahi mau palena ma nā cell mua a me nā cell.

Loaʻa i ka CRISPR/Cas9-mediated ka knockout gene crispr a me ka knockin gene crispr. ʻO ka knockout gene Crispr e pili ana i ka hoʻolauna ʻana i ka ʻoki ʻana o ke kaula pālua o ka DNA, e hopena ana i ka nalowale o ka hana o ka gene i manaʻo ʻia. ʻO ka knockin gene Crispr e pili ana i nā ʻāpana DNA haole i loko o nā genes kikoʻī, hiki ke hoʻohana ʻia e hoʻoponopono i nā genes hemahema a i ʻole e hoʻolauna i nā genes hana hou.

ʻO ka nānā ʻana o ka plasmid ka papahana kūkulu laina cell mua loa, pono wale nō e hoʻololi pono i ka plasmid target i loko o ke kelepona a hoʻohana i ke kū'ē kūpono i ka nānā ʻana i ka wā lōʻihi. A laila kahi manawa kūpono e loaʻa ai nā laina cell paʻa.

Nā ʻano hana o ke kūkulu ʻana i nā laina cell paʻa like ʻole

Ke ʻAno Hoʻoponopono	ʻŌnaehana Lentivirus	ʻŌnaehana Transposon	Hoʻopili ʻia e CRISP/Cas9	Nānā ʻana i ka Plasmid
Ka Mana o ka Mea Lawe	5 kb	10 kb	5 kb	10 kb
Ka Hana Kūpono i Hoʻopili ʻia	Kiʻekiʻe	Maʻamau	Maʻamau	Haʻahaʻa
Ka Papa Manawa	1-2 pule	3-4 pule	3-4 pule	4-6 mau pule
Pōmaikaʻi	Wikiwiki a me ka pono	Palekana a me ke kūpaʻa	Hoʻohui kiko paʻa	Kumukūʻai haʻahaʻa
Ka hemahema	Ka pilikia palekana olaola	Ka nui o ka hōʻike ʻana maʻamau	He paʻakikī a he pipiʻi hoʻi ka hoʻololi ʻana o CAS9	Ka hana haʻahaʻa a me ka paʻa ʻole

Nā pilikia loea i ke kūkulu ʻana i nā laina kelepona paʻa

Ma mua o ka hoʻonui ʻana i ke kaʻina hana, hoʻohana ʻia kahi bioreactor liʻiliʻi e hoʻoholo i nā palena e like me ke kinoea, ka ʻōnaehana wai moʻomeheu, ka pilikia hydrodynamic liʻiliʻi i hoʻopili ʻia i nā cell, a pēlā aku, e hōʻoia i ka moʻomeheu cross-scale. Eia nō naʻe, inā ua hoʻoholo ʻia nā palena hana e pili ana, e like ʻole ke kaiapuni o nā bioreactors nui i loko o ka mea hana, a he mea maʻalahi ke hāʻule. He nui nā makahiki o ka ʻike o Alpha Lifetech i ka moʻomeheu hoʻonui kaʻina hana, hiki ke pale aku i nā hewa i kekahi ʻano, hoʻemi i nā pilikia, a hoʻomaikaʻi nui i ka helu holomua. I ka manawa like, hōʻoia ʻo Alpha Lifetech i ke kūlike o nā mea maka a me nā ʻāpana media i hoʻohana ʻia, e hoʻemi ana i ka helu hāʻule o ka hoʻonui kaʻina hana moʻomeheu cell.

Loaʻa iā Alpha Lifetech kahi kahua hoʻomohala hana laina kelepona paʻa piha e hōʻoia i ke kūpaʻa o nā laina kelepona i ka wā o ka hana ʻana. Hiki iā mākou ke hāʻawi iā ʻoe i nā lawelawe i hoʻopilikino ʻia e hoʻokō i kāu mau pono.

ʻIkepili o ka lawelawe kūkulu ʻana i nā laina kelepona paʻa

ʻIkepili	Ka Papa Manawa
ʻO ka laina kelepona Culture HEK293 i hāʻawi ʻia e ka mea kūʻai aku.	3-5 nā mahina
Ke kūkulu ʻana o ka plasmid sgRNA-Cas9 (me ka gene screening G418).
Ua hoʻoili pū ʻia ka sgRNA-Cas9 a me ka hoʻoponopono DNA template i ka laina cell HEK293.
Ka nānā ʻana i ka laina kelepona monoclonal (gene GFP ke kīkēkē ʻana i ke clone maikaʻi).
Hoʻouna: Vector, ʻano knock-in gene GFP, nā hopena sequencing, nā ʻikepili maka āpau, hōʻike lab

Hiki ke hahai ʻia nā pūnaewele

Ua kūʻai ʻia nā pūnaewele mai nā hui ʻoihana e like me ATCC a hiki ke hāʻawi i nā hōʻike e hoʻokō ai i kāu mau koi hoʻopaʻa inoa.

Ka pōmaikaʻi hana

He nui nā makahiki o ka ʻike o Alpha Lifetech e hoʻokō i nā pono hoʻomohala like ʻole; Loaʻa iā mākou nā ʻano cell like ʻole, kahi hale hana mālama kūʻokoʻa, ka hoʻopaʻa ʻana o nā cell kūpono, a me kahi ʻōnaehana hoʻokele mālama.

ka mana maikaʻi o ka papahana

Hoʻomaʻamaʻa ʻia kēlā me kēia papahana i nā kaʻina hana hoʻomalu maikaʻi e hōʻoia i ka lawe ʻana o nā laina kelepona e kū ana i nā kūlana kiʻekiʻe i nā mea kūʻai aku.

Pōmaikaʻi loea

Ma waho aʻe o nā ʻenekinia me ka ʻike nui o ke kelepona, loaʻa iā mākou nā algorithms evolutionary ʻenehana holomua e hoʻomaikaʻi i nā kūlana reactor a hoʻololi i nā palena hana.

Inā he mau nīnau kāu, e ʻoluʻolu e hoʻokaʻaʻike mai iā mākou i kēlā me kēia manawa.

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