Lecture 17 - Genetic Manipulation in Embryonic Stem Cells & Animals PDF

MIC115 Recombinant DNA Cloning 24FQ Lecture 17– Genetic manipulation in embryonic stem cells and animals Totipotency, pluripotency, and germline biology Text with gray bars: you do not need to remember...

MIC115 Recombinant DNA Cloning 24FQ Lecture 17– Genetic manipulation in embryonic stem cells and animals Totipotency, pluripotency, and germline biology Text with gray bars: you do not need to remember SUGGESTED VIDEOS Blastocyst Development - Day 3 to Day 5 (MUST SEE) https://www.youtube.com/watch?v=uCn1PQP2yAo What are embryonic stem cells? Narrated by Dr. Janet Rossant https://www.youtube.com/watch?v=EY5CTGI03uM The Basics of Crispr-Cas9: Knockout Mice https://www.youtube.com/watch?v=d6aNhPgtCTE&t=18s This is a good video for the last lecture (CRISPR): CRISPR: Gene editing and beyond https://www.youtube.com/watch?v=4YKFw2KZA5o LECTURE 17 TOPICS Embryonic stem (ES) cells Genetically modified animal models CRISPR experiments in ES cells Introduction to mouse genetics LECTURE 17 LEARNING GOALS Understand the basic features of ES cells Describe how CRISPR-mediated genome editing works in ES cells Describe how CRISPR-mediated genome editing works to generate genetically modified animal models Compare knockout, knock-in, vs. transgenic mice I. Embryonic stem (ES) cells ES cells can be used as tools to make genetically modified animals ES cells are derived from the inner cell mass of the blastocyst ES cells have the potential to give rise to all cell types in the body of the organism. These cells can be cultured like an established cell line. (DNA can be introduced by transfection or viral infection/transduction, and the transformed cells can be selected using selectable markers.) 1 MIC115 Recombinant DNA Cloning 24FQ Blastocyst: The outer layer consists of cells collectively called the trophoblast, which gives rise placenta. The inner cell mass is the source of embryonic stem cells capable of forming all cell types within the embryo. Stem cell definition: Self-renewal refers to the ability to go through cycles of cell division while maintaining the undifferentiated state. Differentiation refers to the ability to differentiate into specialized cell types under certain conditions. II. Epigenetic reprogramming and stem cells From pluripotent stem cells, differentiation usually accompanies gain of epigenetic modifications. Q. How can you make pluripotent cells? A. Induction of Induced pluripotent cells (iPS cells). Induction of iPS cells can be done by forced expression of certain transcription factors. Accompanies the loss of epigenetic modifications Reprogramming usually accompanies the loss of epigenetic modifications (and X-inactivation in females). III. Totipotency and pluripotency Totipotency: the ability of a single cell to divide and produce all of the differentiated cells in an organism. ES cells and induced pluripotent stem cells have pluripotency Pluripotency: the potential to give rise to all cell types in the body of an adult organism True or False questions: Q. ES cells are required for normal development. A: False (ES cells is not a part of normal developmental process) Q: ES cells can give rise to all cell types in the body of an adult organism. A: True 2 MIC115 Recombinant DNA Cloning 24FQ Cleavage divisions in the mammalian embryo: Until eight-cell stage embryo: Each cell is totipotent to give rise to a complete animal when implanted into the uterus of a pseudo-pregnant animal. IV. ES cell isolation and culture Human or mouse blastocysts – grown from cleavage-stage embryos produced by in vitro fertilization Inner cell mass separated from surrounding extraembryonic tissues plated onto a layer of fibroblast feeder cells. Feeder cells have been treated with chemical or irradiation, so they won’t proliferate but remain metabolically active to provide specific protein hormones/growth factors for the ES cells. Individual cells – form ES cell colonies – can be maintained for many generations/stored frozen ES cells can give rise to every cell type of the organism If the altered ES cells are injected into a recipient wildtype blastocyst which then implant into foster mother mouse, the resulting baby mouse will be a chimera containing parts derived from the normal mouse parent cells and parts derived from the ES cells! In chimera mice (mosaic), germ cells can be mosaic (ES cell -origin and host-origin) Chimera mice (mosaic) can be mated with wild-type mice. Then, we can obtain pups with 100% modified ES cells origin (germline transmission). Once you make a genetically modified mouse and if it is fertile, you can maintain as a mouse line. V. Conventional gene targeting in mice ES cell targeting - inject targeted ES cells to blactocyst - embryo transfer - founder chimeric mice - germline transmission (Alternatively, directly modify ES cells with CRISPR) But, now you can make a knock out mice in one step. 3 MIC115 Recombinant DNA Cloning 24FQ Direct microinjection of the sgRNA and CAS9 protein complex into cytoplasm of a mouse zygote. - Embryo transfer VI. CRISPR experiments in ES cells Basically, the strategy is as same as that for other cells (described in the previous lecture). CRISPR experiments in ES cells Many Applications: In vitro stem cell studies In vitro differentiation Organogenesis in vitro: Organoids Stem cell therapy VII. Introduction to mouse genetics knockout, Knock-in, vs. Transgenic mice knockout: Inactivation of an existing gene by disrupting it or replacing it with an artificial piece of DNA. Knock-in: One-for-one substitution of DNA sequence information in a genetic locus CRISPR is common Direct microinjection of the sgRNA and CAS9 protein complex into cytoplasm of a mouse zygote. Transgenic mice: Mice that have DNA from another source in the genome. Most popular method: random integration of DNA to genome. Direct microinjection of DNA into the male pronucleus of a mouse zygote. knockout: we mainly examine homozygous mutants in which both parental alleles are deleted (- /-). Heterozygous mutants (+/-) sometime show phenotypes (haploinsufficiency), but most mutations are recessive (+/- does not show phenotypes). Knock-in: Loss-of-function: we mainly examine homozygous mutants (KI/KI). Gain-of-function: heterozygous knock-in mice (KI/+) are sufficient. Transgenic mice: heterozygous transgenic mice (Tg/+) are sufficient. Transgenic mice: random integration of DNA to genome Direct microinjection of DNA into the male pronucleus of a mouse zygote Male pronucleus is large! 4 MIC115 Recombinant DNA Cloning 24FQ An example: A CRISPR knock-in mouse line: A point mutation in which tyrosine 142 (Y142) of histone variant H2AX is converted to alanine (H2ax-Y142A). https://www.sciencedirect.com/science/article/pii/S0960982219315660?via%3Dihub Form a complex with in vitro transcribed sgRNA and CAS9 proteins + a doner DNA oligo (Knock-in allele) Microinjections of the mix into the cytoplasm of fertilized eggs Doner DNA oligo was incorporated to genome by HR On the same day as the microinjections, the eggs were transferred into the oviduct ampulla of pseudopregnant wild-type female mice. 5