B6-Ntcptm1(hNTCP)小鼠,动物试验

NTCP小鼠是通过CRISPR-Cas9技术对在C57BL/6J背景插入人源化的NTCP基因而获得纯合突变小鼠。

粗蛋白≥23.0%、粗脂肪≥5.0%、粗灰分≤7.0%、粗纤维≤5.0%、水份≤10%、钙1.4%～1.5%、磷1.0%～1.1%、赖氨酸≥1.32%、蛋氨酸+胱氨酸≥0.78%

1. 生长曲线

2、解剖学特性与背景品系C57BL/6J相同。3、繁殖特性纯合子之间同胞兄妹间繁殖，繁殖能力一般，每胎产5-7只。4、自发异常

不规则脾 脾脏黑色素沉积

1、NTCP小鼠的制备 背景小鼠C57BL/6J为近交系小鼠，是经过全基因组测序的小鼠，具有实验结果精度高、可比性好、应激反应均一等特点，是基因工程小鼠常用品系。 基因设计信息 NTCP的信息及研究背景 Slc10a1 solute carrier family 10 (sodium/bile acid cotransporter family), member 1 [ Mus musculus (house mouse) ]

Gene ID: 20493

该基因完整的CDNA序列如下：>hNTCP (1050bp)ATGGAGGCCCACAACGCGTCTGCCCCATTCAACTTCACCCTGCCACCCAACTTTGGC

AAGCGCCCCACAGACCTGGCACTGAGCGTCATCCTGGTGTTCATGTTGTTCTTCATC

ATGCTCTCGCTGGGCTGCACCATGGAGTTCAGCAAGATCAAGGCTCACTTATGGAAG

CCTAAAGGGCTGGCCATCGCCCTGGTGGCACAGTATGGCATCATGCCCCTCACGGCC

TTTGTGCTGGGCAAGGTCTTCCGGCTGAAGAACATTGAGGCACTGGCCATCTTGGTC

TGTGGCTGCTCACCTGGAGGGAACCTGTCCAATGTCTTCAGTCTGGCCATGAAGGGG

GACATGAACCTCAGCATTGTGATGACCACCTGCTCCACCTTCTGTGCCCTTGGCATGA

TGCCTCTCCTCCTGTACATCTACTCCAGGGGGATCTATGATGGGGACCTGAAGGACAA

GGTGCCCTATAAAGGCATCGTGATATCACTGGTCCTGGTTCTCATTCCTTGCACCATAG

GGATCGTCCTCAAATCCAAACGGCCACAATACATGCGCTATGTCATCAAGGGAGGGAT

GATCATCATTCTCTTGTGCAGTGTGGCCGTCACAGTTCTCTCTGCCATCAATGTGGGG

AAGAGCATCATGTTTGCCATGACACCACTCTTGATTGCCACCTCCTCCCTGATGCCTTT

TATTGGCTTTCTGCTGGGTTATGTTCTCTCTGCTCTCTTCTGCCTCAATGGACGGTGC

AGACGCACTGTCAGCATGGAGACTGGATGCCAAAATGTCCAACTCTGTTCCACCATCC

TCAATGTGGCCTTTCCACCTGAAGTCATTGGACCACTTTTCTTCTTTCCCCTCCTCTAC

ATGATTTTCCAGCTTGGAGAAGGGCTTCTCCTCATTGCCATATTTTGGTGCTATGAGA

AATTCAAGACTCCCAAGGATAAAACAAAAATGATCTACACAGCTGCCACAACTGAAGAA

ACAATTCCAGGAGCTCTGGGAAATGGCACCTACAAAGGGGAGGACTGCTCCCCTTGCA

CAGCCTAG 1.1 Donor质粒设计及构建质粒 人NTCP基因与小鼠的3’-UTR连接设计donor，形成hNTCP-mUTR，用人源的hNTCP-mUTR替换小鼠基因，形成M-donor-hNTCP-mUTR。

1.2 载体构建 ①sgRNA 载体 载体名称：pUC57-sgRNA expression vector Addgene ID: 51132 根据基因信息选择靶点，合成sgRNA（上海英潍捷基） Targeting site 1GGGTACGAGCTCCACTCGGC AGGM- NTCP-EBe1-gRNA up: TAGGGTACGAGCTCCACTCGGCM- NTCP-EBe1-gRNA down: AAACGCCGAGTGGAGCTCGTAC Targeting site 2GGACCGAGGGTTAAGAGGAA AGGM- NTCP-EAf1-gRNA up: TAGGACCGAGGGTTAAGAGGAAM- NTCP-EAf1-gRNA down: AAACTTCCTCTTAACCCTCGGT合成的sgRNA单链通过退火复性结合成小片段，插入BSAⅠ线性化的载体sgRNA插入位点示意图

构建完成的sgRNA载体通过体外转录成为可注射的sgRNA。（体外转录用试剂盒：Ambion Am1354） ②doner设计>hNTCP (1050bp)ATGGAGGCCCACAACGCGTCTGCCCCATTCAACTTCACCCTGCCACCCAACTTTGGCAAGCGCCCCACAG

ACCTGGCACTGAGCGTCATCCTGGTGTTCATGTTGTTCTTCATCATGCTCTCGCTGGGCTGCACCATGGA

GTTCAGCAAGATCAAGGCTCACTTATGGAAGCCTAAAGGGCTGGCCATCGCCCTGGTGGCACAGTATGGC

ATCATGCCCCTCACGGCCTTTGTGCTGGGCAAGGTCTTCCGGCTGAAGAACATTGAGGCACTGGCCATCT

TGGTCTGTGGCTGCTCACCTGGAGGGAACCTGTCCAATGTCTTCAGTCTGGCCATGAAGGGGGACATGAA

CCTCAGCATTGTGATGACCACCTGCTCCACCTTCTGTGCCCTTGGCATGATGCCTCTCCTCCTGTACATCT

ACTCCAGGGGGATCTATGATGGGGACCTGAAGGACAAGGTGCCCTATAAAGGCATCGTGATATCACTGGT

CCTGGTTCTCATTCCTTGCACCATAGGGATCGTCCTCAAATCCAAACGGCCACAATACATGCGCTATGTCA

TCAAGGGAGGGATGATCATCATTCTCTTGTGCAGTGTGGCCGTCACAGTTCTCTCTGCCATCAATGTGGG

GAAGAGCATCATGTTTGCCATGACACCACTCTTGATTGCCACCTCCTCCCTGATGCCTTTTATTGGCTTTC

TGCTGGGTTATGTTCTCTCTGCTCTCTTCTGCCTCAATGGACGGTGCAGACGCACTGTCAGCATGGAGAC

TGGATGCCAAAATGTCCAACTCTGTTCCACCATCCTCAATGTGGCCTTTCCACCTGAAGTCATTGGACCAC

TTTTCTTCTTTCCCCTCCTCTACATGATTTTCCAGCTTGGAGAAGGGCTTCTCCTCATTGCCATATTTTGG

TGCTATGAGAAATTCAAGACTCCCAAGGATAAAACAAAAATGATCTACACAGCTGCCACAACTGAAGAAAC

AATTCCAGGAGCTCTGGGAAATGGCACCTACAAAGGGGAGGACTGCTCCCCTTGCACAGCCTAG小鼠的3’-UTRAATGTGAAAACTTAGAAGCAACAAGGAAAGTAACACCTAGTGTGCCAGATTGTCTAGCACTTCCAGCAAAC

CTTCAACAGCAGAATCATGAAGCAATGAACTGAGGCAGAAGGGCATCTATCCAGGAACCATCATCCATCCC

CAAAAATCTGCTATTTGTTTAAAAGATAAAAAAGAACTAGGCAAAGTGGTTCCTCCCTATAATTCCAATGCT

CAGAGACTCAAGGTCAACCTTGAGTACACAGCAAGACTGTCTCAAGAAACCAAGAACACTTTCAGCAGCTA

TGAACTCTTATGAAGGCTCTATGAAACAGCTACATCTGATAAACATTATCACTATTTCTAGACTTTCCAATA

AGCAGGTGTTTTGCTCATTAAGCATCCACAACCTGCTTCATGTTGTAACTCAAAAGGAAATAAACTACAACT

GGTAGTTCTAACTTACCAAAC> hNTCP-mUTRATGGAGGCCCACAACGCGTCTGCCCCATTCAACTTCACCCTGCCACCCAACTTTGGCAAGCGCCCCACAGA

CCTGGCACTGAGCGTCATCCTGGTGTTCATGTTGTTCTTCATCATGCTCTCGCTGGGCTGCACCATGGAGT

TCAGCAAGATCAAGGCTCACTTATGGAAGCCTAAAGGGCTGGCCATCGCCCTGGTGGCACAGTATGGCATC

ATGCCCCTCACGGCCTTTGTGCTGGGCAAGGTCTTCCGGCTGAAGAACATTGAGGCACTGGCCATCTTGGT

CTGTGGCTGCTCACCTGGAGGGAACCTGTCCAATGTCTTCAGTCTGGCCATGAAGGGGGACATGAACCTCA

GCATTGTGATGACCACCTGCTCCACCTTCTGTGCCCTTGGCATGATGCCTCTCCTCCTGTACATCTACTCCA

GGGGGATCTATGATGGGGACCTGAAGGACAAGGTGCCCTATAAAGGCATCGTGATATCACTGGTCCTGGTT

CTCATTCCTTGCACCATAGGGATCGTCCTCAAATCCAAACGGCCACAATACATGCGCTATGTCATCAAGGGA

GGGATGATCATCATTCTCTTGTGCAGTGTGGCCGTCACAGTTCTCTCTGCCATCAATGTGGGGAAGAGCATC

ATGTTTGCCATGACACCACTCTTGATTGCCACCTCCTCCCTGATGCCTTTTATTGGCTTTCTGCTGGGTTATG

TTCTCTCTGCTCTCTTCTGCCTCAATGGACGGTGCAGACGCACTGTCAGCATGGAGACTGGATGCCAAAATG

TCCAACTCTGTTCCACCATCCTCAATGTGGCCTTTCCACCTGAAGTCATTGGACCACTTTTCTTCTTTCCCCTC

CTCTACATGATTTTCCAGCTTGGAGAAGGGCTTCTCCTCATTGCCATATTTTGGTGCTATGAGAAATTCAAGA

CTCCCAAGGATAAAACAAAAATGATCTACACAGCTGCCACAACTGAAGAAACAATTCCAGGAGCTCTGGGAAA

TGGCACCTACAAAGGGGAGGACTGCTCCCCTTGCACAGCCTAGAATGTGAAAACTTAGAAGCAACAAGGAAAG

TAACACCTAGTGTGCCAGATTGTCTAGCACTTCCAGCAAACCTTCAACAGCAGAATCATGAAGCAATGAACTGA

GGCAGAAGGGCATCTATCCAGGAACCATCATCCATCCCCAAAAATCTGCTATTTGTTTAAAAGATAAAAAAGAA

CTAGGCAAAGTGGTTCCTCCCTATAATTCCAATGCTCAGAGACTCAAGGTCAACCTTGAGTACACAGCAAGACT

GTCTCAAGAAACCAAGAACACTTTCAGCAGCTATGAACTCTTATGAAGGCTCTATGAAACAGCTACATCTGATA

AACATTATCACTATTTCTAGACTTTCCAATAAGCAGGTGTTTTGCTCATTAAGCATCCACAACCTGCTTCATGTT

GTAACTCAAAAGGAAATAAACTACAACTGGTAGTTCTAACTTACCAAAC 小鼠基因组序列attattttttttacctggtgcatctcaagcttgctacaacctcacttccttctctgaagtctctcaagttatggatacctcaggcaccagctagttactctc

cacagctggagtttctcgtgagctgggctttggataatctccaggcttccaagagcccaacgtaccccagaaagttttttcccctctggagataagt

aagtctttcagggattcatggtgccaccccccaaccaaccccaacccccacaggacacgtatggtctagatatgggcacaaagcaaggtctcag

aggaggacagtcatggacacagcctctcctgaagacagcaacaccacatcgcccccccatcctgtcccatggccctctggcctttgccacttaga

tgaggaaggcaaaggcagaaacagtctctttctctcgggaacaaatggtgactcactgaggatagggaaggcaccttttcccaggaaacaacc

tgaggtctccgagactgagctagaagcaggaactaaagggaaaggtggacatgttttgttgcaggtcctggatgggtctcagagacgctatag

tgaagcatgctcagcagggtaaagaatgaaccctgtgctgggtacgagctccactcggcaggcacagaggtcagaaaacttaagcaaagtag

gggatccggggagatgaggttatgggctgtcttataaccacaaccgtggttctgcaggttgcATAATCTTTTATTTGCCCACAGCTAC

ATTTTGTCCAGAAACTCTGTCCTGAAAGAGGACTGAAGAAAACATCCAGCCAGCACTGGGCAAGGAAGGGCAG

GAGCAGAGAGTGAGGTGGTGTTCACTGGGTCGGAGGATGGAGGCGCACAACGTATCAGCCCCCTTCAATTTC

TCCCTGCCGCCTGGCTTTGGCCACCGGGCCACAGACACTGCGCTCAGCGTCATTCTGGTAGTTATGTTGCTGC

TCATCATGCTCTCGCTTGGCTGCACCATGGAGTTCAGCAAGATCAAGGCTCACTTCTGGAAGCCCAAAGGGGT

GATCATCGCCATAGTGGCCCAGTACGGTATCATGCCCCTCAGTGCTTTCCTTCTGGGCAAGGTCTTTCATCTG

ACCAGCATTGAGGCTCTGGCCATCCTCATCTGCGGCTGCTCTCCTGGGGGGAACCTGTCTAACCTCTTCACCC

TGGCCATGAAGGGGGACATGAACCTCAGgtaggaccgagggttaagaggaaagggtgtgggatggggtgctgccgagatgtca

tgatctagagaagatctaggctggagccaggagcggggagggacgtaggagctggattaagtttggcattgattgtcaaaggtttatgctaaa

ttggggcagcctctggaatctctttggtttaaatcccagggggaaaaaaaaaacattaagctggtccagaggtatgttagacacccctgaactc

ccagaagtctctacaggatcactgtaaaggagaatagatttcgatgaaagagttcctcagaaccacagtcatctgactggcagggtgtcagct

ctgaggctgaaaatctagcaagggtataatgggtccagaacattccaggaaggccaaggtgacttggaaccaaacgagcatcttaaatttcc

tgcagagaagagactccaaaggctcttctccagtatctgggcatccaaggtatctgctaagaccctttaccgagttgtcagatgtggtgagatt

gccaaccaaagaaactttatgtagaattgaggccccagcgattgcccaggttaggctgggctccactcagtctaacaagaagggagaagg

ctaagaccagcctgagcagccaggtggtggtggtgcatgcctttaatcccagcacttgggaggcagaggcaggtggatttctgagttgaggc

cagcctggtctacaaagtgagttccaggacagtcaggactataca 用人源的hNTCP-mUTR替换上述下滑线序列，包含一处点突变，命名为M-donor-hNTCP-mUTR>M-donor-hNTCP-mUTRAttattttttttacctggtgcatctcaagcttgctacaacctcacttccttctctgaagtctctcaagttatggatacctcaggcaccagctagttactct

ccacagctggagtttctcgtgagctgggctttggataatctccaggcttccaagagcccaacgtaccccagaaagttttttcccctctggagataa

gtaagtctttcagggattcatggtgccaccccccaaccaaccccaacccccacaggacacgtatggtctagatatgggcacaaagcaaggtctc

agaggaggacagtcatggacacagcctctcctgaagacagcaacaccacatcgcccccccatcctgtcccatggccctctggcctttgccactta

gatgaggaaggcaaaggcagaaacagtctctttctctcgggaacaaatggtgactcactgaggatagggaaggcaccttttcccaggaaaca

acctgaggtctccgagactgagctagaagcaggaactaaagggaaaggtggacatgttttgttgcaggtcctggatgggtctcagagacgcta

tagtgaagcatgctcagcagggtaaagaatgaaccctgtgctgggtacgagctccactcggcagg(T)cacagaggtcagaaaacttaagca

aagtaggggatccggggagatgaggttatgggctgtcttataaccacaaccgtggttctgcaggttgcATAATCTTTTATTTGCCCACA

GCTACATTTTGTCCAGAAACTCTGTCCTGAAAGAGGACTGAAGAAAACATCCAGCCAGCACTGGGCAAGGAAG

GGCAGGAGCAGAGAGTGAGGTGGTGTTCACTGGGTCGGAGGATGGAGGCCCACAACGCGTCTGCCCCATTCA

ACTTCACCCTGCCACCCAACTTTGGCAAGCGCCCCACAGACCTGGCACTGAGCGTCATCCTGGTGTTCATGTTG

TTCTTCATCATGCTCTCGCTGGGCTGCACCATGGAGTTCAGCAAGATCAAGGCTCACTTATGGAAGCCTAAAGG

GCTGGCCATCGCCCTGGTGGCACAGTATGGCATCATGCCCCTCACGGCCTTTGTGCTGGGCAAGGTCTTCCGG

CTGAAGAACATTGAGGCACTGGCCATCTTGGTCTGTGGCTGCTCACCTGGAGGGAACCTGTCCAATGTCTTCA

GTCTGGCCATGAAGGGGGACATGAACCTCAGCATTGTGATGACCACCTGCTCCACCTTCTGTGCCCTTGGCAT

GATGCCTCTCCTCCTGTACATCTACTCCAGGGGGATCTATGATGGGGACCTGAAGGACAAGGTGCCCTATAAA

GGCATCGTGATATCACTGGTCCTGGTTCTCATTCCTTGCACCATAGGGATCGTCCTCAAATCCAAACGGCCACA

ATACATGCGCTATGTCATCAAGGGAGGGATGATCATCATTCTCTTGTGCAGTGTGGCCGTCACAGTTCTCTCTG

CCATCAATGTGGGGAAGAGCATCATGTTTGCCATGACACCACTCTTGATTGCCACCTCCTCCCTGATGCCTTTTA

TTGGCTTTCTGCTGGGTTATGTTCTCTCTGCTCTCTTCTGCCTCAATGGACGGTGCAGACGCACTGTCAGCATG

GAGACTGGATGCCAAAATGTCCAACTCTGTTCCACCATCCTCAATGTGGCCTTTCCACCTGAAGTCATTGGACC

ACTTTTCTTCTTTCCCCTCCTCTACATGATTTTCCAGCTTGGAGAAGGGCTTCTCCTCATTGCCATATTTTGGTG

CTATGAGAAATTCAAGACTCCCAAGGATAAAACAAAAATGATCTACACAGCTGCCACAACTGAAGAAACAATTCC

AGGAGCTCTGGGAAATGGCACCTACAAAGGGGAGGACTGCTCCCCTTGCACAGCCTAGAATGTGAAAACTTAGA

AGCAACAAGGAAAGTAACACCTAGTGTGCCAGATTGTCTAGCACTTCCAGCAAACCTTCAACAGCAGAATCATGA

AGCAATGAACTGAGGCAGAAGGGCATCTATCCAGGAACCATCATCCATCCCCAAAAATCTGCTATTTGTTTAAAA

GATAAAAAAGAACTAGGCAAAGTGGTTCCTCCCTATAATTCCAATGCTCAGAGACTCAAGGTCAACCTTGAGTAC

ACAGCAAGACTGTCTCAAGAAACCAAGAACACTTTCAGCAGCTATGAACTCTTATGAAGGCTCTATGAAACAGCT

ACATCTGATAAACATTATCACTATTTCTAGACTTTCCAATAAGCAGGTGTTTTGCTCATTAAGCATCCACAACCTG

CTTCATGTTGTAACTCAAAAGGAAATAAACTACAACTGGTAGTTCTAACTTACCAAACgaaagggtgtgggatggggtg

ctgccgagatgtcatgatctagagaagatctaggctggagccaggagcggggagggacgtaggagctggattaagtttggcattgattgtcaaa

ggtttatgctaaattggggcagcctctggaatctctttggtttaaatcccagggggaaaaaaaaaacattaagctggtccagaggtatgttagacac

ccctgaactcccagaagtctctacaggatcactgtaaaggagaatagatttcgatgaaagagttcctcagaaccacagtcatctgactggcagggtg

tcagctctgaggctgaaaatctagcaagggtataatgggtccagaacattccaggaaggccaaggtgacttggaaccaaacgagcatcttaaattt

cctgcagagaagagactccaaaggctcttctccagtatctgggcatccaaggtatctgctaagaccctttaccgagttgtcagatgtggtgagattgc

caaccaaagaaactttatgtagaattgaggccccagcgattgcccaggttaggctgggctccactcagtctaacaagaagggagaaggctaagac

cagcctgagcagccaggtggtggtggtgcatgcctttaatcccagcacttgggaggcagaggcaggtggatttctgagttgaggccagcctggtct

acaaagtgagttccaggacagtcaggactataca ③ CAS9 载体载体名称：pST1374-NLS-flag-linker-Cas9Addgene ID: 44758载体通过体外转录成为可注射的Cas9-RNA（体外转录用试剂盒：Ambion Am1345） 1.3显微注射①超数排卵：150只3-4周龄c57雌鼠注射激素进行超排。②受精卵注射：取约350枚受精卵进行注射。③雄鼠结扎：制作30只8周龄输精管结扎的c57雄鼠。④受体鼠制备：8-10周龄ICR雌鼠与结扎的ICR雄鼠交配后选取见栓的雌鼠。⑤胚胎移植：将注射后的受精卵移植到受体鼠输卵管壶腹部（移植一次， 350枚卵，15只受体鼠）。