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    Cell modification method using essential genes as markers and optionally recycling these
    2.
    发明授权
    Cell modification method using essential genes as markers and optionally recycling these 有权

    公开(公告)号：US09951339B2

    公开(公告)日：2018-04-24

    申请号：US14427698

    申请日：2013-09-19

    Applicant: DSM IP ASSETS B.V.

    Inventor： Ilse De Lange ,  Bernard Meijrink ,  Johannes Andries Roubos ,  Siew-loon Ooi

    IPC: C12N15/65 ,  C12N15/80 ,  C12N15/81

    CPC classification number: C12N15/65 ,  C12N15/80 ,  C12N15/81

    Abstract: The invention relates to a method for modification of a host cell at a target locus, which method comprises: providing a host cell comprising, at a first locus, at least two site-specific recombination sites and a nucleic acid having an essential function or encoding a product having an essential function; introducing into the host cell, at the target locus, a further nucleic acid having the essential function or encoding for a product having the essential function; and carrying out recombination at the first locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function is rendered non-functional, thereby to modify the host cell at the target locus. The invention also relates to a cell obtainable by a method of the invention.

    POLYPEPTIDE EXPRESSION METHOD
    4.
    发明申请
    POLYPEPTIDE EXPRESSION METHOD 审中-公开
    多肽表达方法

    公开(公告)号：US20150064765A1

    公开(公告)日：2015-03-05

    申请号：US14394771

    申请日：2013-04-23

    Applicant: DSM IP ASSETS B.V.

    Inventor： Johannes Andries Roubos ,  Jan Metske Van Der Laan ,  Bastiaan Van Den Berg ,  Dick De Ridder

    IPC: C12N9/42 ,  C12N9/30

    CPC classification number: C12N9/2437 ,  C12N9/242 ,  C12N15/67 ,  C12P21/02 ,  C12Y302/01001 ,  C12Y302/01004

    Abstract: A method for the production of a polypeptide of interest in a host cell, which method comprises: a. providing a host cell which harbours a nucleic acid encoding a polypeptide of interest, wherein the polypeptide of interest is modified so that it comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence; b. cultivating the host cell under conditions suitable for production of the polypeptide; and, optionally, c. recovering the compound of interest. The invention also relates to a modified polypeptide which comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence.

    Abstract translation: 一种在宿主细胞中产生感兴趣的多肽的方法，该方法包括：a。 提供宿主细胞，其含有编码感兴趣的多肽的核酸，其中所述目的多肽被修饰，使得其包含比参考多肽更少的甲硫氨酸和/或赖氨酸残基，不包括位于N端的任何初始甲硫氨酸氨基酸 多肽序列的末端; b。 在适于生产多肽的条件下培养宿主细胞; 和可选地，c。 回收目的化合物。 本发明还涉及一种修饰的多肽，其包含比参考多肽更少的甲硫氨酸和/或赖氨酸残基，不包括位于多肽序列的N-末端的任何初始甲硫氨酸氨基酸。

    Nucleic Acid Assembly System
    7.
    发明申请
    Nucleic Acid Assembly System 审中-公开
    核酸装配系统

    公开(公告)号：US20140303036A1

    公开(公告)日：2014-10-09

    申请号：US14359358

    申请日：2012-11-23

    Applicant: DSM IP ASSETS B.V.

    Inventor： Johannes Andries Roubos ,  Bernard Meijrink ,  Richard Kerkman ,  Ben Den Dulk

    IPC: C12N15/10

    CPC classification number: C12N15/1082 ,  C12N15/1027 ,  C12N15/1093 ,  C12N15/52 ,  C12N15/80 ,  C12N15/905

    Abstract: The present invention relates to a method for the preparation of a library of host cells, a plurality of which comprise an assembled polynucleotide at a target locus, which method comprises: (a) providing a plurality of polynucleotides comprising two or more polynucleotide subgroups, wherein: (i) a plurality of polynucleotides in each polynucleotide subgroup comprises sequence encoding a peptide or polypeptide and/or a regulatory sequence; (ii) a plurality of peptides or polypeptides encoded by, or a plurality of regulatory sequences comprised within, each polynucleotide subgroup share an activity and/or function; (iii) at least one polynucleotide subgroup comprises at least two non-identical polynucleotide species; (iv) a plurality of polynucleotides of each polynucleotide subgroup comprises sequence enabling homologous recombination with a plurality of polynucleotides from one or more other polynucleotide subgroups; and (v) a plurality of polynucleotides in two polynucleotide subgroups comprise a nucleotide sequence enabling homologous recombination with a target locus in host cells; and (b) assembling the plurality of polynucleotides at the target locus by homologous recombination in vivo in host cells, thereby to generate a library of host cells, a plurality of which comprise an assembled polynucleotide at the target locus. The assembled polynucleotides may be recovered, thereby to prepare a library of nucleic acids.

    Abstract translation: 本发明涉及一种用于制备宿主细胞文库的方法，其多个在靶位点包含组装的多核苷酸，该方法包括：（a）提供多个包含两个或更多个多核苷酸亚基的多核苷酸，其中 （i）每个多核苷酸亚组中的多个多核苷酸包含编码肽或多肽和/或调节序列的序列; （ii）每个多核苷酸亚组中由多个或多个编码的多肽或多肽调节序列共享活性和/或功能; （iii）至少一个多核苷酸亚组包含至少两个不相同的多核苷酸种类; （iv）每个多核苷酸亚组的多个多核苷酸包括能够与来自一个或多个其它多核苷酸亚组的多个多核苷酸进行同源重组的序列; 和（v）两个多核苷酸亚组中的多个多核苷酸包含能够与宿主细胞中的靶基因座进行同源重组的核苷酸序列; 和（b）通过在宿主细胞中的体内同源重组在靶位点组装多个多核苷酸，从而产生宿主细胞文库，其多个包含靶标位点处的组装的多核苷酸。 可以回收组装的多核苷酸，从而制备核酸文库。

    Assembly system for a eukaryotic cell
    8.
    发明授权
    Assembly system for a eukaryotic cell 有权

    公开(公告)号：US11149268B2

    公开(公告)日：2021-10-19

    申请号：US16319746

    申请日：2016-12-15

    Applicant: DSM IP ASSETS B.V.

    Inventor： Johannes Andries Roubos ,  René Verwaal ,  Bianca Elisabeth Maria Gielesen ,  Brenda Vonk

    IPC: C12N15/09 ,  C12N15/10 ,  C12N15/11 ,  C12N15/90

    Abstract: The present invention is based on the advantageous use of single-stranded oligonucleotides in the in vivo (within a cell) assembly of double-stranded oligonucleotides into a single double-stranded nucleic acid construct.
    The present invention relates to the use of at least a first and a second single-stranded oligonucleotide in the assembly within a cell of at least two double-stranded nucleic acid molecules into a single double-stranded nucleic acid construct of pre-determined sequence, wherein the first and second single-stranded oligonucleotide are essentially complementary to each other.

    CELL MODIFICATION METHOD USING ESSENTIAL GENES AS MARKERS AND OPTIONALLY RECYCLING THESE
    9.
    发明申请
    CELL MODIFICATION METHOD USING ESSENTIAL GENES AS MARKERS AND OPTIONALLY RECYCLING THESE 有权
    使用基因基因作为标记的细胞修饰方法和可循环的这些方法

    公开(公告)号：US20150225728A1

    公开(公告)日：2015-08-13

    申请号：US14427698

    申请日：2013-09-19

    Applicant: DSM IP ASSETS B.V.

    Inventor： Ilse De Lange ,  Bernard Meijrink ,  Johannes Andries Roubos ,  Siew-loon Ooi

    IPC: C12N15/65 ,  C12N15/80

    CPC classification number: C12N15/65 ,  C12N15/80 ,  C12N15/81

    Abstract: The invention relates to a method for modification of a host cell at a target locus, which method comprises: providing a host cell comprising, at a first locus, at least two site-specific recombination sites and a nucleic acid having an essential function or encoding a product having an essential function; introducing into the host cell, at the target locus, a further nucleic acid having the essential function or encoding for a product having the essential function; and carrying out recombination at the first locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function is rendered non-functional, thereby to modify the host cell at the target locus. The invention also relates to a cell obtainable by a method of the invention.

    Abstract translation: 本发明涉及用于在靶标位点修饰宿主细胞的方法，该方法包括：提供宿主细胞，其在第一场所包含至少两个位点特异性重组位点和具有基本功能或编码的核酸 具有基本功能的产品; 在靶标位点向宿主细胞中引入具有基本功能或编码具有基本功能的产品的另一种核酸; 并通过至少两个位点特异性重组位点在第一位点进行重组，使得具有基本功能或编码具有基本功能的产物的核酸变得不起作用，从而修饰宿主细胞 目标位点。 本发明还涉及可通过本发明的方法获得的细胞。

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