SSRs and Population Genetics Notes

SSRs and Population Genetics Notes - 7.03 Genetics –...

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Unformatted text preview: 7.03 Genetics – Lectures 28-32 TA: Yun Song 912-484-2511 | yuns2010@mit.edu Review – SSRs and Population Genetics 3 3 2. the most 18 is one of the most common human populations. You in human populations. You prepare Trisomy 18 is oneTrisomy 18 is one trisomies observed in trisomies observed in human populations. You of Trisomy common of the most common trisomies observed prepare DNA samples from two unrelated infants,and from their both and from pare DNA samples from samples from two unrelated infants, bothwith trisomy 18, 18, and their their parents. You then DNA two unrelated infants, both with trisomy 18, with trisomy from parents. then type the infants and parents for four SSRs distributed ents. You then type thetheYouand their parents parentstheir four SSRs distributed along along type infants infants and their for four SSRs distributed along for chromosome 18: chromosome 18: omosome 18: SSR1 5 cM S SR2 SSR1 1. SSRs and Chromosomal Anomalies S SR2 SSR3 5 cM 40cM S SR4 SSR3 S SR4 10 cM 40 cM 10 cM Family 1 A B C A B C A B C A B C Family 1 A B C A B C A B C A B C S SR1 S SR1 S SR2 S SR2 S SR3 S SR3 S SR4 S SR4 In which parent did(a) In which parent did nondisjunction occur in Family 1? nondisjunction occur in Family 1? In which division of b) In which division of meiosis didin Family 1? ( meiosis did nondisjunction occur nondisjunction occur in Family 1? (a) In which parent did nondisjunction occur in Family 1? (b) In which division of meiosis did nondisjunction occur in Family 1? Meiosis 2 (c) Sketch the meiotic event in which nondisjunction occurred in Family 1. Your drawing should include the SSRs present along chromosome 18. Sketch the meioticmotherwhichmeiotic event inoccurred in Family 1. occurred in Family 1. Your drawing should (event in the nondisjunction which nondisjunction Your drawing should c) Sketch ude the SSRs present along chromosome 13. include the SSRs present along chromosome 13. 7.03 Genetics – Lectures 28-32 TA: Yun Song 912-484-2511 | yuns2010@mit.edu (continued). 4 Family 2 A B C A B A B C A B C S SR1 S SR2 C S SR3 S SR4 ) In which parent did nondisjunction occur in Family 2? ) In which division of meiosis did nondisjunction occur in Family 2? (d) In which parent did nondisjunction occur in Family 2? father Family 2? Meiosis I (f) Sketch the meiotic event in which nondisjunction occurred. Your drawing should include the SSRs present along chromosome 18. Sketch the meiotic event in which nondisjunction occurred. Your drawing should include the Rs present along (e) In which division of meiosis did nondisjunction occur in chromosome 13. Population Genetics Trekking in the Himalayas, you discover a “founder generation” of 1000 goats barricaded on all sides by high peaks and massive glaciers. This founder generation consists of 200 AA goats, 200 Aa goats, and 600 aa goats. (a) What are the frequencies of A and a alleles in the founder generation? f(A) = p = f(AA) + ½ f(Aa) = 0.2 + 0.1 = 0.3 f(a) = q = f(aa) + ½ f(Aa) = 0.6 + 0.1 = 0.7 (b) Is the founder generation in Hardy-Weinberg Equilibrium? p2 = 0.32 = 0.09 ≠ f(AA) No, they are not in HW equilibrium. 7.03 Genetics – Lectures 28-32 TA: Yun Song 912-484-2511 | yuns2010@mit.edu (c) What is f(A) in the second generation? (Mating of the founder generation goats is random, fitness does not differ among the three genotypes, and mutation occurs at a negligible rate.) f(A) does not change. f(A) = 0.3 (d) What are the frequencies of AA, Aa, and aa genotypes in the second generation? f(AA) = p2 = 0.09 f(Aa) = 2pq = 0.42 f(aa) = q2 = 0.49 (e) Let’s now suppose that a new disease arises in the goat population. Goats with the genotype aa are especially susceptible, while AA and Aa goats are unaffected. If waa=0.5 and wAA=wAa=1, what are the new allele frequencies in the next generation (two generations after the founder generation)? Mean wA = 1 p' = p (wA/w) = 0.3 (1/0.755) = 0.397 Mean wa = 0.65 q’ = q (wa/w) = 0.7 (0.75/0.755) = 0.603 Mean w = 0.755 (f) Assuming this type of selection and a (A a) mutation rate of 10-6, what will be the allele frequencies at equilibrium? peq will be ~ 1 qeq = sqrt(µ/s) Since waa = 0.5, s = 0.5. qeq = sqrt(10-6 / 0.5) = 1.4 x 10-3 Main Concepts Transgenesis Purpose: insert gain of function allele DNA Construct: Promoter Gene X ORF OR via Transposon Gene X Transposase + Type of cell DNA added to: germ cells of animal or fertilized egg (pronucleus injection) 7.03 Genetics – Lectures 28-32 TA: Yun Song 912-484-2511 | yuns2010@mit.edu Type of integration into genome: random Chimera generated? No Additional breeding required? - If germ cell: mate with homozygous recessive animal - If fertilized egg: no Gene-Targeting – “Knock-Out” Purpose: create loss of function allele DNA Construct: -------------NeoR ------------ TkNSV Type of cell DNA added to: ESC Type of integration into genome: homologous recombination Chimera generated? Yes Additional breeding required? P0: chimera X WT black mouse (KO/+; Aw/Aw) (+/+; a/a) (+/+; a/a) F1: non-chimeric agouti ½ (KO/+; Aw/a) ½ (+/+; Aw/a) x sibling F2: ¼ (KO/KO) 7.03 Genetics – Lectures 28-32 TA: Yun Song 912-484-2511 | yuns2010@mit.edu Tissue-Specific “KO” Purpose: generate knock-outs in certain tissues, esp useful for embryonic lethal gene deletions Reagent needed: CRE recombinanse Recombination site: loxP DNA constructs: 1) --->>>YFG>>>--How was this generated? Knock-in of YFG + loxP sites 2) TissueSpecific Promoter CRE How was this generated? Random insertion by transgenesis Strains of mice we need to cross: --->>>YFG>>>----->>>YFG>>>--X TissueSpecific Promoter TissueSpecific Promoter CRE CRE --->>>YFG>>>--- ; Specific Tissue- Promoter CRE X Sibling ¼ (YFG/YFG), ¾ (CRE/?) ¼ x ¾ = 3/16 useful mice Types of tissues we will see in final mouse generated: --->>> YFG >>>----->>> YFG >>>--Promoter OFF – no CRE And --->>>----->>>--Promoter ON – CRE expressed, YFG deleted 7.03 Genetics – Lectures 28-32 TA: Yun Song 912-484-2511 | yuns2010@mit.edu Question Based on Lecture Many mouse genes are “tissue-specific,” that is, they are present throughout the body but are expressed in only one of the animal’s many tissue types. (Other mouse genes are expressed throughout the body, or in multiple tissues.) Geneticists can study the regulation of a mouse gene by fusing the gene’s promoter region to the LacZ coding sequence and injecting the construct to create a transgenic mouse. Fusion of the mouse amylase promoter to LacZ yielded a Pamylase-LacZ construct. (a) Would microinjection of the Pamylase-LacZ construct into the male pronucleus of a fertilized egg likely result in integration of the construct into the amylase gene? Briefly explain your answer. The construct will insert randomly into the genome (homologous insertion is unlikely since only Pamylase shows homology to any sequence on the genome). Random insertion into the amylase gene is extremely unlikely. (b) Mice heterozygous for the resulting Pamylase-LacZ transgene displayed LacZ expression exclusively in the pancreas. Would you expect homozygotes for the transgene to also display LacZ expression in the pancreas? Elsewhere? Briefly explain your answer. The insert has a dominant phenotype, so a homozygote for the transgene will also show the same phenotype – LacZ will only be expressed in the pancreas. Regulation of the amylase gene should be same in the heterozygote and the homozygote. However, expression in other organs is also possible in the homozygote if expression of 7.03 Problem Sets Fall 2003 7.03 Fall but one copy of LacZ is just below the level of detection,2003 expression of two copies is detectable. to amylase upstream and downstream sequences. The re (c) You are surprised to observe that mice homozygous (knock-in) mice (after ES cells targeting, blastocyst inje for the transgene insertion display a serious heart defect. (Heterozygotes have normal hearts.) Suggest a possible explanation. mating of chimeras to wild-type mice) can be assayed to The transgene probably inserted into a gene necessary certainly do heart function (gene not for normal so. D). This loss of function in one copy of the gene does not affect the animal, but when both copies are lost, we see the heart defect. in the pancreas. The expression of LacZ is likely to refl (e) Propose how you might use LacZ in a gene targeting experiment in mice to test whether the amylase gene is expressed exclusively in the pancreas. Draw a labeled diagram of the targeting construct that you would use, and outline any breeding experiments required to test your hypothesis. 1. Construct a DNA sequence to knock out gene D – DNA construct should contain LacZ flanked by regions homologous to gene D and Tk. ----Tk 2. Transfect (black) ES cells with DNA construct. Select for LacZ-expressing and GanR cells (these contain the construct in the desired region). 3. Inject ES cells into (white, recessive) blastocyst and generate chimeric mice. nstream sequences. The resulting heterozygous knock-out 7.03 Genetics – Lectures 28-32 lls targeting, blastocyst Song injection to generate chimera, and TA: Yun ype mice) can be assayed to |determine if LacZ is expressed 912-484-2511 yuns2010@mit.edu ion of LacZ is likely to reflect amylase expression, but will 4. Breed chimera (black+white) with recessive white mice to generate mice heterozygous for LacZ knock-in. 5. Assay organs for X-gal staining to determine if LacZ (and thus amylase) is only expressed in the pancreas. 6 ...
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This note was uploaded on 02/06/2009 for the course 7 7.03 taught by Professor Fink during the Spring '09 term at MIT.

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